Chimeric adeno-associated virus/ bocavirus parvovirus vector

ABSTRACT

The invention provides an isolated chimeric virus comprising bocavirus capsid protein and a recombinant adeno-associated viral (AAV) genome, an isolated rBoV comprising human bocavirus capsid protein and a recombinant BoV genome, and uses therefor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/782,876, filed Oct. 7, 2015, which application is a U.S. NationalStage Application under 35 U.S.C. 371 from International Application No.PCT/US2014/033343, filed Apr. 8, 2014, which application claims thebenefit of the filing date of U.S. application Ser. No. 61/809,702 filedon Apr. 8, 2013, the disclosures of which are incorporated herein.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under HL108902 awardedby National Institutes of Health. The Government has certain rights inthe invention.

BACKGROUND

Gene therapy has been widely used in clinical trials since 1990s withmany successful cases reported using viral or non-viral vectors todeliver therapeutic genes. The lung is an important organ for the genetherapy treatment to patients with inherent gene defects such as cysticfibrosis (CF), alpha 1-antitrypsin (AAT) deficiency, or with otherchronic acquired respiratory disorders such as asthma and lung cancers.Of these lung diseases, CF, caused by single gene defect in coding aprotein cystic fibrosis transmembrane conductance regulator (CFTR), isthe most common life-threatening gene defect inherent disease with about$450 million spent annually on patient care in the U.S. alone. Althoughclinical treatments have improved CF patients' quality of life andlifespan in the recent decades, for this single gene defect inherentdisease, gene therapy appears the best cure to permanently correct thedisorder by replacing the defective CFTR gene (Mueller et al., 2008;Driskell et al., 2003; Griesenbach et al., 2010).

CF is an autosomal recessive genetic disorder caused by mutations in theCFTR gene coding (Rommens et al., 1989). It is a multi-organ disease,but CF pulmonary disease is the most life-threatening (Rowe et al.,2005). Recombinant adeno-associated viral vectors (rAAV) are currentlyone gene therapy agent that is being pursued for CF lung gene therapy(Griesenbach et al., 2010; Flotte, 2007; Carter, 2005).

rAAV vectors for CF lung gene therapy have been under development fornearly two decades, and most serotypes appear to be effectivelyendocytosed from the apical surface of airway epithelia despite varyingdegrees of transduction (i.e., expression of an encoded transgene).Although these vectors have demonstrated good safety profiles in CFclinical trails (Aitken et al., 2001; Moss et al., 2007; Wagner et al.,2002), they have failed to achieve complementation in vivo for twosignificant reasons. First, post-entry barriers in virion processingfollowing infection appear to limit nuclear translocation, and thustransgene expression, in a proteasome-dependent manner (Duan et al.,2000; Ding et al., 2005; Yan et al., 2002; Zhong et al., 2008; Zhong etal., 2007). This feature of rAAV2 is reflected in CF clinical trialswhere viral genomes persisted in the airway epithelia of test subjectswithout detection of transgene-derived CFTR mRNA or clinical improvementin lung function (Aitken et al., 2001; Moss et al., 2007; Wagner et al.,2002). Identifying an appropriate rAAV serotype that bypassed theselimitations has proved challenging due to species-specific differencesbetween animal models and humans (Flotte et al., 2010; Liu et al.,2007a; Liu et al., 2007b). rAAV1 proves to be the most efficientserotype for apical infection of human airway (Flotte et al., 2010; Yanet al., 2012; Yan et al., 2006), while others have found success usingdirected capsid evolution to enhanced the tropism of rAAV for apicalhuman epithelium (HAE) transduction (Li et al., 2009; Excoffon et al.,2009). However, effective CFTR complementation in CF HAE still requiresthe use of proteasome inhibitors to enhance transduction (Li et al.,2009; Zhang et al., 2004).

A second major barrier to efficient CFTR expression from rAAV vectors istheir limited packaging capacity (about 4.9 kb) that necessitates theuse of small, weak promoters and/or the use of CFTR minigenes (Zhang etal., 1998). The first generation rAAV-CFTR tested in a clinical trialutilized the cryptic promoter within the AAV2 ITR to drive theexpression of a full-length CFTR cDNA (Aitken et al., 2001), and thiswas later improved by the incorporation of a short 83 bp syntheticpromoter (Zhang et al., 2004). Other efforts to circumvent the smallpacking capacity of rAAV vectors have included trimming down size of theCFTR cDNA by deletion of non-critical sequences (such as partialdeletion at the R-domain) to expand room for core promoter elements suchas a shortened CMV promoter (Li et al., 2009; Zhang et al., 1998;Ostedgaard et al., 2005; Ostedgaard et al., 2002). Although thesestrategies have improved expression of CFTR, it is clear that pushingthe packaging limits of rAAV can lead to inconsistent deletions at the5′ end of rAAV genome (Kapranov et al., 2012), thus further jeopardizinggenome stability and expression.

SUMMARY OF THE INVENTION

As described herein, a recombinant human bocavirus virus-1 (HBoV-1) wasgenerated from an ORF-disrupted rHBoV1 genome that efficientlytransduces human airway epithelium (HAE) from the apical surface. Thelarger genome and high airway tropism of HBoV1 is ideal for creating aviral vector for gene transfer, e.g., airway gene transfer, includinggene therapy for genetic and acquired diseases such as genetic andacquired pulmonary diseases, cancer, as well as vaccines, for instance,against respiratory disease. As further described herein, a rAAV2/HBoV1chimeric virus (e.g., about 5.5-kb genome) was created, where HBoV1capsids packaged oversized rAAV2 genomes. Clinical trials have supportedthe safety of applying the rAAV2 genome in the context of gene therapyfor cystic fibrosis (CF) lung disease. The chimeric vector retains thehigh safety profile of the rAAV2 genome while also providing the airwayapical tropism of the HBoV1 capsid. rAAV2/HBoV1 was shown to be capableof apically transducing HAE at 5.6- and 70-fold greater efficiency thanrAAV1 or rAAV2 (4.7-kb genomes), respectively. Molecular studiesdemonstrated that polarization of airway epithelial cells was requiredfor HBoV1 capsid-mediated gene transfer. Further, rAAV2/HBoV1-CFTR viruscontaining the full-length CFTR coding sequence and the strong CBApromoter efficiently corrected CFTR-dependent chloride transport incystic fibrosis HAE. Thus, the chimeric AAV/HBoV viral vector is usefulfor gene therapy of cystic fibrosis and other pulmonary diseases, andthe development of vaccines against HBoV1 infections and otherrespiratory viruses such as influenza virus. Co-administration ofproteasome inhibitors during the infection period also significantlyenhanced the AAV/HBoV1 chimeric vector transduction by a thousand fold.

The invention thus provides a gene transfer vector. e.g., for humanpulmonary disease gene therapy and vaccines. The vector is highly tropicfor the human airway, has spacious package capacity of the HBoV capsid,and efficiently encapsidates the rAAV genome. As a highly efficientairway transduction vector, the vector may be employed for CF genetherapy strategies, as well as gene therapy for other pulmonary diseasessuch as AAT deficiency, chronic obstructive pulmonary disease (COPD),asthma, lung cancers, as well as vaccination against wild-type HBoVinfections and other respiratory infections (such as influenza virus andrespiratory syncytial virus (RSV) infections), e.g., in infants,toddlers, juveniles or adults.

The invention also provides a platform for the development of bocavirus(BoV)-based gene transfer vaccines with rAAV genomes for use in humans,pets, and livestock, including but not limited to pulmonary diseases.The bocavirus capsid for the gene transfer vectors, e.g. recombinantbocavirus vector (rBoV) and chimeric adeno-associated/bocavirusparvoviral vector (rAAV/BoV), can be from different stains of humanbocaviruses and non-human bocaviruses. Human bocavirus 1 (HBoV1) is arespiratory virus of tropism to infect the airway tract and humanbocavirus 2 to 4 (HBoV2, HBoV3 and HBoV4) are enteric viruses of tropsimto infect the gastrointestinal tract. Non-human bocaviruses, such asswine bocavirus, canine bocavirus and feline bocavirus, are isolatedfrom non-human mammals.

In one embodiment, the invention provides an isolated chimeric viruscomprising a bocavirus capsid protein, e.g., a human bocavirus capsidprotein, and a recombinant heterologous parvovirus genome, e.g., arecombinant adeno-associated viral (AAV) genome. For example, the rAAVgenome may include an expression cassette encoding a heterologous geneproduct, e.g., which is a therapeutic protein such as cystic fibrosistransmembrane conductance regulator, α-antitrypsin, β-globin, γ-globin,tyrosine hydroxylase, glucocerebrosidase, aryl sulfatase A, factor VIII,dystrophin or erythropoietines an antigen such as viral, bacterial,tumor or fungal antigen, or a neutralizing antibody or a fragmentthereof that targets an epitope of an antigen such as one from a humanrespiratory virus, e.g., influenza virus or RSV. In one embodiment, thegene product is a therapeutic gene product. In one embodiment, the geneproduct is a prophylactic gene product. In one embodiment, the geneproduct is a catalytic RNA. In one embodiment the gene product is apolypeptide or peptide. In one embodiment, the capsid protein is HBoV1,HBoV2, HBoV3 or HBoV4 capsid protein. In one embodiment the bocaviruscapsid protein is from a bocavirus isolated from a non-human speciesthat imparts a unique tropism for infection of lung or other organs, forexample, porcine bocavirus. In one embodiment, the rAAV/HBoV or rAAV/BoVvector used for vaccination is used in animals to protect lifestock orpets. In one embodiment, the AAV genome is an AAV-1, AAV-2 or AAV-5genome. In one embodiment, the MV genome is a AAV-1, AAV-3, AAV-4,AAV-5, AAV-6, AAV-7, AAV-8 or AAV-9 genome.

BoV sequences within the scope of the invention include but are notlimited to nucleic acid sequences having at least 80%, 85%, 90%, 95%,98%, 99% or 100% nucleic acid sequence identity to contiguous sequenceshaving, for example, one of SEQ ID Nos. 9, 17-18, 39, or 42-43, or thecomplement thereof. BoV capsid sequences within the scope of theinvention include but are not limited to amino acid sequences having atleast 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to sequences having,for example, one of SEQ ID Nos. 21-24, 39-41, or 44-45.

The invention provides a method of preparing a chimeric virus comprisinga bocavirus (BoV) capsid protein and a recombinant heterologousparvovirus genome, such as a recombinant AAV (rAAV) genome. The methodincludes providing a first vector comprising a nucleic acid sequence fora recombinant MV genome; a second vector comprising a nucleic acidsequence for one or more adenovirus genes for MV replication, forinstance, one or more of the E4orf6 gene, the E2A protein gene, and theVA RNA genes; a third vector comprising a nucleic acid sequence encodingone or more Rep proteins, e.g., Rep40, Rep52, Rep68 or Rep78; and afourth vector comprising a terminal sequence that is a deleted bocavirusgenome that encodes BoV1 capsid and gene product(s) for encapsidation.Cells, e.g., mammalian or insect cells, are transfected with the vectorsin an amount effective to yield the chimeric virus. In one embodiment,the vectors for introduction to insect cells include a AAV2 Rep helperbaculovirus (Bac-AAV2Rep), which expresses AAV2 Rep78/Rep52, a HBoV1 Caphelper virus (Bac-HBoVCap), which expresses HBoV1 capsid proteins VP1,VPx, and VP2; and a transfer vector (Bac-rAAV), which contains an rAAV2genome carrying gene of interest (GOI). The insect cells are infectedwith these baculovirus vectors in an amount effective to yield thechimeric virus.

In one embodiment, the chimeric virus may not include a transgene, buthas ITRs and a non-coding sequence (“stuffer” sequence). Such a virushas a capsid (e.g., a HBoV capsid) that induces a humoral response andso is useful as a vaccine. In one embodiment, the chimeric virus isdelivered to the lungs. In one embodiment, the chimeric virus isdelivered to the nose, tracheobronchial airways and/or lungs. In oneembodiment, the chimeric virus is generated with BoV strains that infectother organs, such as the gastrointestinal tract. In one embodiment, thechimeric virus is used to infect humans. In one embodiment, the chimericvirus is used to infect animals such as livestock or pets.

In one embodiment, the chimeric virus includes a transgene, the geneproduct of which enhances humoral or cellular response to BoV and hasITRs. Such a virus is useful as a vaccine as a result of the humoralresponse to the BoV capsid and the immune response (humoral and/orcellular) that is enhanced by expression of the transgene. In oneembodiment, the chimeric virus is delivered to the lungs. In oneembodiment, the chimeric virus is delivered to the nose,tracheobronchial airways and/or lungs. In one embodiment, the chimericvirus is generated with BoV strains that infect other organs. In oneembodiment, the chimeric virus is used to infect humans. In oneembodiment, the chimeric virus is used to infect animals such aslivestock or pets.

In one embodiment, the chimeric AAV/BoV virus includes a transgene andhas ITRs. The transgene may encode any antigen, e.g., a tumor antigen,BoV proteins (but not proteins that allow for BoV replication),influenza virus protein, e.g., H1 or N1 protein, or SARS viral genessuch as capsid genes), or an immune response modulator, e.g., cytokinesincluding but not limited to IFN-alpha, IFN-gamma, TNF, IL-1, IL-17, orIL-6, or other gene products that enhance the cellular or humoral immuneresponse. In one embodiment, the chimeric virus is delivered to thelungs. In one embodiment, the chimeric virus is delivered to the nose,tracheobronchial airways and/or lungs. In one embodiment, the chimericvirus is generated with BoV strains that infect other organs. In oneembodiment, the chimeric virus is used to infect humans. In oneembodiment, the chimeric virus is used to infect animals such aslivestock or pets.

In one embodiment, the transgene may encode an antibody for passiveimmunization, for instance, against respiratory virus infections, e.g. abroadly neutralizing antibody targeted the epitopes conserved amongdiverse influenza virus strains, or against other respiratory virusessuch as respiratory syncytial virus (RSV) and SARS virus. In oneembodiment, the chimeric virus is generated with BoV strains that infectorgans other than the respiratory tract. In one embodiment, the chimericvirus is used to infect humans. In one embodiment, the chimeric virus isused to infect animals such as livestock or pets

Further provided is a method to enhance chimeric virus transduction of amammalian cell. The method includes contacting a mammalian cell, e.g., ahuman cell, with an isolated chimeric virus comprising bocavirus capsidprotein and a rAAV genome encoding a heterologous gene product and atleast one agent in an amount effective to additively or synergisticallyenhance rAAV transduction. In one embodiment, the mammalian cell is amammalian lung cell. In one embodiment, the agent is a chemotherapeutic,a lipid lowering agent, a mucolytic agent, an antibiotic or a foodadditive. In one embodiment, the mammalian cell is a mammalian cellother than the lung for which alternative strains of bocavirus (isolatedfrom human or other animals) allow for efficient infection. In oneembodiment, the agent is a proteasome modulator, e.g., a proteasomeinhibitor.

The invention includes a method to enhance virus transduction of amammalian cell, e.g., a mammalian lung cell. For example, a mammalianlung cell is contacted with a chimeric virus comprising a bocaviruscapsid protein and a rAAV genome and an agent in an amount effective toenhance transduction of the virus relative to a mammalian cell that isnot contacted with the agent, wherein the agent is a proteasomeinhibitor.

In one embodiment, the invention provides a method to enhance theexpression of a transgene in a mammalian cell, such as a mammalian lungcell. The method includes contacting the mammalian cell with an amountof an agent that is a proteasome inhibitor and a chimeric viruscomprising a human bocavirus capsid protein and a rAAV genome comprisingthe transgene, wherein the amount enhances transduction of the rAAV,thereby enhancing expression of the transgene, relative to a mammaliancell that is not contacted with the agent.

In one embodiment, the invention provides a method to immunize a mammal.The method includes contacting a mammal with a chimeric virus comprisinga bocavirus capsid protein and a recombinant heterologous parvovirusgenome, e.g., rAAV genome, comprising a transgene useful to induce aprotective immune response to an antigen, e.g., a microbial antigen suchas a virus, bacteria, parasite, or fungus, or a tumor antigen, or aneutralizing antibody or fragment thereof useful to prevent infectionsby a pathogen including but not limited to a virus, bacterium, fungus orparasite. In one embodiment, the mammal is also contacted with aproteasome inhibitor. In one embodiment, the transgene encodes aneutralizing antibody or an antigen binding fragment thereof. Thus, thechimeric virus may be employed as a vaccine, e.g., a passive vaccine.

Also provided is a method to inhibit or treat a condition associatedwith aberrant expression of an endogenous gene product. The methodincludes contacting a mammal at risk of or having the condition, with aneffective amount of at least one proteasome inhibitor, achemotherapeutic, a lipid lowering agent, a mucolytic agent, anantibiotic or a food additive that enhances transduction and aneffective amount of an isolated chimeric virus comprising bocaviruscapsid protein and a rAAV genome, wherein the genome comprises atransgene encoding at least a portion of a functional gene product, theexpression of which in the mammal inhibits or treats at least onesymptom of the condition. In one embodiment, the transgene encodescystic fibrosis transmembrane conductance regulator, alpha-1antitrypsin, β-globin, γ-globin, tyrosine hydroxylase,glucocerebrosidase, aryl sulfatase A, factor VIII, dystrophin orerythropoietin.

In one embodiment, a mammal subjected to viral gene therapy with anisolated chimeric virus comprising bocavirus capsid proteins and a rAAVgenome is administered an agent that is a proteasome inhibitor in anamount effective to enhance expression of a transgene in the rAAV in thecells of the mammal relative to cells in a mammal that are not contactedwith the agent.

Further provided is a rHBoV virus. In one embodiment, the rHBoV virusmay not include a transgene, but has terminal palindromic sequences(TPSs) that are not identical and a non-coding sequence (“stuffer”sequence), i.e., it is not replication competent. Such a virus has acapsid (BoV) that induces a humoral response and so is useful as avaccine. In one embodiment, the chimeric virus is delivered to thelungs. In one embodiment, the chimeric virus is delivered to othernon-lung cell types for which BoV capsid sequences are tropic forinfection.

To produce rBoV, in one embodiment, two or more vectors are employed.One vector has cis elements for replication and packaging, which includethe TPSs, and optionally a heterologous sequence (transgene). The othervector has sequences for trans acting factors but lacks the cis elements(they are deleted). The two vectors may be on one plasmid or twodifferent plasmids. Moreover, the trans acting factors may be ondifferent plasmids. For example, sequences for the non-structuralproteins, e.g., NS and NP1, may be on one plasmid and another plasmidmay have sequences for the capsid proteins. Structural proteins requiredfor packaging rBoV may also be split into multiple vectors to avoidgeneration of wild-type BoV.

In one embodiment the AAV/BoV virus is produced in cultured insectcells. This method includes the utility of recombinant baculovirusvectors (BEV): a MV Rep helper baculovirus (Bac-AAVRep), which expressesAAV Rep78/Rep52, a BoV1 Cap helper virus (Bac-BoVCap), which expressesBoV1 capsid proteins VP1, VPx, and VP2; and a transfer vector(Bac-rAAV), which contains an rAAV genome carrying gene of interest(GOI). The insect cells are infected with these baculovirus vectors inan amount effective to yield the chimeric virus.

In one embodiment, the rBoV virus includes a transgene, the gene productof which enhances humoral or cellular response to BoV and has TPSs,e.g., it is not by itself replication competent or can produceinfectious BoV. Such a virus is useful as a vaccine as a result of thehumoral response to the BoV capsid and the immune response (humoraland/or cellular) that is enhanced by expression of the transgene. In oneembodiment, the rHBoV is delivered to the lungs. Structural proteinsrequired for packaging rBoV may also be split into multiple vectors toavoid generation of wild-type BoV. In one embodiment, the chimeric virusis delivered to other non-lung cell types for which BoV capsid sequencesare tropic for infection

In one embodiment the AAV/BoV virus is produced in cultured insectcells. This method includes the utility of recombinant baculovirusvectors (BEV): a AAV Rep helper baculovirus (Bac-AAVRep), whichexpresses AAV Rep78/Rep52, a BoV1 Cap helper virus (Bac-BoVCap), whichexpresses BoV1 capsid proteins VP1, VPx, and VP2; and a transfer vector(Bac-rAAV), which contains an rAAV genome carrying gene of interest(GOI). The insect cells are infected with these baculovirus vectors inan amount effective to yield the chimeric virus.

In one embodiment, the rHBoV virus includes a transgene and has HBoVTPSs. The transgene may encode any antigen, e.g., a tumor antigen, HBoVproteins (but not proteins that allow for HBoV replication), influenzavirus protein, e.g., H1 or N1 protein, or SARS viral genes such ascapsid genes)), or an immune response modulator, e.g., a cytokineincluding but not limited to IFN-alpha, IFN-gamma, TNF, IL-1, IL-17, orIL-6 or other gene products that enhance the cellular or humoral immuneresponse. In one embodiment, the rBoV is delivered to the nose,tracheobronchial airways and/or lungs. In one embodiment, the vector forvirus production includes the TPSs and NS sequences, and replaces thecapsid sequences with the transgene, which allows for replication incells but without other sequences provided in trans, does not generateprogeny. In one embodiment, the vector for virus production includes theTPSs and NS sequences, and replaces the capsid sequences with atransgene for a prodrug for tumor cells or a cytokine, e.g., IFN-alpha,IFN-gamma, IL-1, TNF, or IL-17, to enhance the immune response to BoV.

Further provided is a method to enhance rBoV transduction of a mammaliancell. The method includes contacting a mammalian cell, e.g., a humancell, with an isolated rHBoV comprising bocavirus capsid protein and arBoV genome encoding a heterologous gene product and in one embodimentincludes at least one agent in an amount effective to additively orsynergistically enhance transduction. In one embodiment, the mammaliancell is a mammalian lung cell. In one embodiment, the agent is aproteasome modulator, e.g., a proteasome inhibitor. In one embodiment,the agent is a chemotherapeutic, a lipid lowering agent, an antibiotic,a mucolytic agent, or a food additive.

The invention includes a method to enhance virus transduction of amammalian cell, e.g., a mammalian lung cell. For example, a mammalianlung cell is contacted with a rBoV comprising a bocavirus capsid proteinand a rBoV genome and optionally an agent in an amount effective toenhance transduction of the virus relative to a mammalian cell that isnot contacted with the agent, wherein the agent is a proteasomeinhibitor.

In one embodiment, the invention provides a method to enhance theexpression of a transgene in a mammalian cell, such as a mammalian lungcell. The method includes contacting the mammalian cell with an amountof an agent that is a proteasome inhibitor and a rBoV comprising abocavirus capsid protein and a rBoV genome comprising the transgene,wherein the amount enhances transduction of the rBoV, thereby enhancingexpression of the transgene, relative to a mammalian cell that is notcontacted with the agent.

In one embodiment, the invention provides a method to immunize a mammal.The method includes contacting a mammal with a rBoV comprising abocavirus capsid protein, e.g., a human bocavirus capsid protein, and arBoV genome comprising a transgene useful to induce a protective immuneresponse to an antigen, e.g., a microbial antigen such as a virus,bacteria, parasite, or fungus, or a tumor antigen, or a neutralizingantibody or an antigen binding fragment thereof. In one embodiment, themammal is also contacted with a proteasome inhibitor. Thus, the rBoV maybe employed as a vaccine, e.g., a passive vaccine.

Also provided is a method to inhibit or treat a condition associatedwith aberrant expression of an endogenous gene product. The methodincludes contacting a mammal at risk of or having the condition, with aneffective amount of at least one proteasome inhibitor, achemotherapeutic, a lipid lowering agent, an antibiotic, a mucolyticagent, or a food additive that enhances transduction and an effectiveamount of an isolated rBoV comprising human bocavirus capsid protein anda rBoV genome, wherein the genome comprises a transgene encoding atleast a portion of a functional gene product, the expression of which inthe mammal inhibits or treats at least one symptom of the condition. Inone embodiment, the transgene encodes cystic fibrosis transmembraneconductance regulator, alpha1-antitrypsin, β globin, γ-globin, tyrosinehydroxylase, glucocerebrosidase, aryl sulfatase A, factor VIII,dystrophin or erythropoietin.

In one embodiment, a mammal subjected to viral gene therapy with anisolated rHBoV comprising human bocavirus capsid proteins and a rHBoVgenome is administered an agent that is a proteasome inhibitor in anamount effective to enhance expression of a transgene in the rHBoVgenome the cells of the mammal relative to cells in a mammal that arenot contacted with the agent.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-E. rHBoV1 vector production and infection of primary polarizedHAE. (A) Schematic structure of the HBoV1 genome and the proviralplasmids used in this study. pHBoV1KUm630 is the helper plasmid fortrans-complementation of HBoV1 viral proteins, pIHBoV1 is the infectiousclone of the HBoV1 complete genome, and prHBoV1-CBAluc is the rHBoV1 cistransfer proviral plasmid. Critical restriction enzyme cutting sitesused for cloning are also indicated and small deletions within NS and VPgenes are marked (Δ). (B) Replication complementation assay of therHBoV1 proviral plasmid in HEK293 cells. pIHBoV1 (lane 1),prHBoV1-CBAluc (lane 2), or prHBoV1-CBAluc+prHBoV1KUm630 (lane 3)plasmids were transfected to HEK293 cells. Hirt DNA was extracted at 48hours post-transfection and digested by DpnI before resolving on anagarose gel. HBoV1 replication intermediates (indicated by arrows) werevisualized by Southern blotting with a ³²P-labeled HBoV1 probe. Hirt DNAfrom cells transfected with the HBoV1 infectious clone (pIHBoV1, lane 1)was used as positive control. Short and long exposures are shown on theleft and right of the panel, respectively. (C) DNase I-digested celllysates from HEK293 co-transfected with rHBoV1-CBAluc and prHBoV1KUm630were fractionated by CsCl equilibrium ultracentrifugation. The plotshows the distribution of rHBoV1.CBAluc (solid dots) genomes against theobserved density of the gradient (open dots). The genome copies in eachfraction (about 750 μL) were determined by TaqMan PCR. (D) Transductionassay following rHBoV1.CBAluc infection of HEK293 cells, IB3 cells,undifferentiated (UD) CuFi8 cell monolayers, polarized CuFi8 cells inALI cultures, and primary HAE ALI cultures. Data represents the mean(+/−SEM) relative luciferase activity per well at 2-day post-infection(n=4). (E) Transgene expression from rHBoV1.CBAluc infected HAE ALIcultures at different time points post-infection. Data represents themean (+/−SEM) relative luciferase activity per well (n=3).

FIGS. 2A-D. Pseudopackaging rAAV2 genomes in HBoV1 capsid. (A) DNaseI-digested cell lysates from the indicated HEK293 cell plasmidtransfections were fractionated by CsCl equilibrium ultracentrifugation.The number of viral genomes in each fraction was determined by TaqManPCR. (B) HEK293 cells were transfected with the indicated combinationsof plasmids (M: Molecular weight marker; lane 1:pAV2-F5tg83luc+pAV-Rep2; lane 2: pAV2-F5tg83luc+pAVRC2.3; lane 3:pAV2-CF5tg83luc+pAV-Rep2; land 4: pAV2-CF5tg83+pAV-Rep2+pHBoV1KUm630)together with the Ad helper pAd4.1. Low molecular weight (Hirt) DNA wasextracted from transfected cells after 48 hours and digested with DpnI,followed by Southern blotting using a ³²P-labeled luciferase probe. The4.8 kb and 5.4 kb replicative form (RF) DNA of the rAV2.F5tg83Luc andrAV2.CF5tg83Luc genomes are indicated by arrows. (C) Negatively stainedtransmission electron micrographs of the chimeric vectorrAV2/HBc.F5tg83luc (bar=100 nm in the 15000× image and 50 nm for the50000× image). The virus-like particle with incompletely packaged viralDNA (<1% of total virions) is marked by a white arrow in the inset. (D)A two-color Western blot (Red: AAV; Green: HBoV1) was performed on theindicated viral preparations using an Infrared Image System. Convertedsingle channel images are also shown with dark arrows pointing to theAAV2 and HBoV1 VP proteins (VP1 and VP2) in the left and right panels,respectively. Grey arrows and white arrows mark protein from HBoV1 VPxproteins.

FIGS. 3A-C. Package polarity and capacity of rHBoV1 and rAAV2/HBoV1vectors. (A) Viruses AV2/2.F5tg83luc, AV2/HBc.F5tg83luc, andrHBoV1.CBAluc were loaded on nylon membrane by slot blotting andvisualized with ³²P-labeled 32-mer oligonucleotide probes against theminus and plus strand of the Luciferase gene (left panels). Thepercentages of the minus and plus strands in each viral preparation wascalculated based on the signal density quantitated with NIH ImageJsoftware (right panel). (B) 2×10⁸ DRP of rAAV vector AV2/2.F5tg83luc,chimeric viruses AV2/HBc.F5tg83luc, and AV2/HBc.CF5tg83luc were heatedin alkaline gel loading buffer at 95° C. for 10 minutes and thenresolved in a 0.9% alkaline agarose gel. Following transferred to Nylonmembrane, Southern blotting was performed with ³²P-labeled Luciferaseprobe. Black and white arrows mark the shorter rAV2.F5tg83luc (4.8 kb)and longer rAV2.CF5tg83luc (5.4 kb) genomes, respectively. (C) Leftpanels depict slot blots of AV2/HBc.F5tg83luc and rHBoV1.CBAluc viralpreparations (about 10⁹ DRP based on TaqMan PCR for the luciferasetransgene) probed with ³²P-labeled fragments recognizing the luciferasegene (1.7 kb) or the HBoV1 genome region unique to the helper plasmid (a2.64 kb HindIII/BglII fragment covering the NP1 coding region). Rightpanel depicts the relative copies of luciferase or NP1 gene fragmentsbased on the signal intensity relative to the plasmid standards. NIHImageJ software was used to quantify the mean (+/−range) signal densityfor rAAV2/HBoV1 and rHBoV1 viral preparations shown.

FIGS. 4A-D. Transduction comparisons between rAAV2/HBoV1 and rAAVvectors. (A) Luciferase expression at 2 days following infection ofHEK293 cells with AV2/2.F5tg83luc (MOI=2,500 DRP/cell) orAV2/HBc.F5tg83luc (MOI=50,000 DRP/cell). Results show the mean (+/−SEM,N=4) relative luciferase activities per well of a 24-well plate. (B)Primary HAE ALI cultures were infected with AV2/2.F5tg83luc,AV2/1.F5tg83luc, or AV2/HBc.F5tg83luc from the apical or basolateralsurface. The vector amount in the inoculum was 10¹⁰ DRP for eachMillicell insert, roughly 5,000 to 10,000 DRP/cell. Data represent themean (+/−SEM) relative luciferase activities measured at 7 dayspost-infection (RLU/well) for N=6 independent infections of HAE ALIcultures derived from three donors. (C, D) Virion internalization andsubcellular distribution analyses were performed at 18 hours afterprimary HAE ALI cultures were apically infected with rAAV2/1, rAAV2/2and rAAV2/HBoV1 vectors of 10¹⁰ DRP per Millicell insert. Viral genomesin the cytoplasmic and nuclear fractions were quantified by TaqMan PCR.The total viral genomes detected in each culture is presented in (C)with the black bars representing the nuclear fraction and while barsrepresenting the cytoplasmic fraction. The percentage of viral genomesin each fraction is presented in (D). Data represent the mean (+/−SEM)viral genome copies (per well) for N=3 independent infections.

FIGS. 5A-C. Effect of proteasome inhibitors on rAAV2/HBoV1 transductionin polarized and nonpolarized cultures of human airway epithelial cells.(A, B) Primary HAE ALI cultures were apically infected with 10¹⁰ DRP perMillicell insert with (A) AV2/2.F5tg83luc or (B) AV2/HBc.F5tg83luc for aperiod of 16 hours. When indicated, proteasome inhibitors (PI) LLnL (40nM) and doxorubicin (5 μM) were applied only during the infectionperiod. Luciferase expression was monitored over 11 days by biophotonicimaging of live cells using the Xenogen 200 IVIS. Data represent themean (+/−SEM, n=6) relative luciferase activity per well at three timepoints of 3, 7 and 11 day post-infection. (C) CuFi8 cells cultured as apolarized epithelium at an ALI (CuFi-ALI; a: apical infection, b:basolateral infection) or non-polarized undifferentiated monolayers onplastic (CuFi-UD), and HEK293 cells, were incubated with 1.5×10⁹ DRP ofAV2/HBc.F5tg83luc at 37° C. for 4 hours. All cultures contained about5×10⁵ cells at the time of infection. Following infection, unbound viruswas washed off and cells were either detached from the culture supportswith trypsin and lysed for TaqMan PCR quantification of viral genomes,or returned to the incubator for luciferase expression assays at 24hours post-infection using cell lysates. When indicated (+PI). CuFi8cells were treated with proteasome inhibitors doxorubicin (1 μM) andLLnL (8 nM) during the 4 hour infection period. Data represent the mean(+/−SEM) total vector genomes (n=4) at 4 hours post-infection andrelative luciferase activity (n=3) at 24 hours post-infection.

FIGS. 6A-C. Partial correction of CFTR-dependent chloride transport byprimary CF HAE ALI cultures following infection with AV2/HBc.CBAhCFTR.CF HAE ALI cultures derived from two CF patient donors (genotypes:ΔF508/ΔF508 homozygous) were infected with AV2/HBc.CF5tg83luc orAV2/HBc.CBAhCFTR at 10¹⁰ DRP per Millicell insert (MOI of 5000 to 10000DRP/cell) in the presence of proteasome inhibitors LLnL (40 nM) anddoxorubicin (5 μM). Uninfected non-CF HAE were also cultured forelectrophysiologic comparisons and experimental cultures were evaluatedat 10 days postinfection. (A) Representative traces of transepithelialshort-circuit current (Isc) of CF HAE following the sequential additionof various inhibitors and agonists as indicated. Amiloride and DIDS wereused to block ENaC-mediated sodium currents and non-CFTR chloridechannels prior to cAMP agonists (forskolin and IBMX) induction andGlyH101 inhibition of CFTR currents. Δlsc_((cAMP)) reflects theactivation of CFTR-mediated chloride currents following cAMP agonistinduction and Δlsc_((glyH)) reflects the inhibition of CFTR-mediatedchloride currents following addition of GlyH101. (B) Summary data of theΔlsc_((cAMP)) and Δlsc_((glyH)) (mean+/−SEM, n=6 independent transwells)for both CF infected cultures and non-CF controls. (C) Immunofluorescentdetection of CFTR expression (green) in CF HAE following infection withAV2/HBc.CBAhCFTR (left panels) or AV2/HBc.CF5tg83luc (right panels).

FIGS. 7A-B. One potential model for how polarization of human airwayepithelia cells influences HBoV1 virion infection and transduction. (A)Polarized HAE may contain multiple binding and/or coreceptors for HBoV1.In this illustrated scenario, a single binding receptor exists on theapical membrane and is significantly reduced or absent on thebasolateral membrane. Two different coreceptors exist including anefficient co-receptor-1 on the apical membrane and a more abundantinefficient co-receptor-2 on the basolateral membrane. Endocytosisthrough co-receptor-1 leads to functionally efficient (from atransduction standpoint) virion processing that is highly influences byactivity of the proteasome, whereas internalization throughco-receptor-2 is ineffective at processing the virion and not influencedby proteasome function. This model is consistent with significantly lessviral uptake and transduction from the basolateral surface, as comparedto the apical membrane. Other models not shown might include a secondtype of binding receptor on the basolateral surface that isinefficiently endocytosed with co-receptor-1 or co-receptor-2. (B) Innon-polarized human airway cells, the primary binding receptor,co-receptor-1, and co-receptor-2 exist in the same membrane. Bothcoreceptors can interact with the same binding receptor, however,co-receptor-2 is in greater abundance than co-receptor-1. Thus,endocytosis of HBoV1 virions through co-receptor-2 predominates, andsince this pathway inefficiently processes HBoV1 virions for productivetransduction, transgene expression is low. These findings are consistentwith high-level HBoV1 virion endocytosis, but poor transduction and weakproteasome inhibitor responsiveness, in non-polarized human airwaycells.

FIG. 8. Exemplary HoBV sequences including a full length nucleotidesequence (JQ923422, with left 5′ hairpin at nts 1-140 and right 3′hairpin at nts 5344-5543, which are the cis elements for HBoV1replication and packaging), nucleotide sequences (e.g., GQ925675)without terminal hairpins at both ends and proteins encoded thereby.Proteins useful in the viruses of the invention include proteins havingat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% amino acidsequence identity to the sequence of the HBoV proteins in FIG. 8. SEQ IDNOs: 9-36.

FIGS. 9A-9C. Optimization of rAAV2/HBoV1 vector production in 293 cells.(A) HBoV1 Cap helper plasmids. (1) pHBoV1NSCap is the prototype HBoV1helper; (2) pCMVHBoV1NSCap was derived from the prototype helper, a CMVpromoter in front of the P5 promoter; and (3) pCMVHBoV1NS1(−)Cap wasderived from pCMVHBoV1NSCap, with the NS1 ORF terminated early. (B)Western blot analysis of HBoV1 VP1, VPx and VP2 in the 4-plasmidtransfected 293 cell production system (transfected with pAV2.CMVGFP(5.4kb), pAd4.1, pAV2-Rep, and one of the Cap helper independent transwells)for both CF infected cultures and non-CF controls. (C) The yield ofrAAV2/HBoV1 from the improved rAAV2/HBoV1 production system (transfectedwith pAV2.CMVGFP(5.4 kb), pAd4.1, pAV2-Rep and pCMVHBoV1NS1(−)Caphelper) was comparable to that of rAAV2/2 production system (transfectedwith pAV2.CMVGFP(5.4 kb), pAd4.1, pAV2-RepCap helper) in 293 cells.Comparison was plotted from side-by-side preparations at the scale of 20145-mm plates.

FIGS. 10A-10E. rAAV2/HBoV1 production in Sf9 cells. (A) Construction ofthe BEV for rAAV2/HBoV1 production. The three BEV shown were generatedusing the Bac-to-Bac method (Invitrogen). Bac-Cap was designed accordingto the Kotin method, but the a silent point mutation was introduced atnt 273 (G to A) of the VP1-coding sequence to ensure an appropriateratio of VP1:VPx:VP2. Bac-Rep was also constructed according to theKotin method; Ph: polyhedrin promoter. (B) Analysis of virus proteinexpression and rAAV2 DNA replication. At 72 hours p.i., the Sf9 cellsinfected with the 3 BEV were analyzed for the expression of HBoV1 Capand AAV2 Rep by Western blotting, and for replication of the rAAV2genome by Southern blotting. HBoV1 Cap proteins (VP1, VPx, and, VP2)were produced efficiently, at a ratio similar to that in pHBoV1NSCap-transfected 293 cells, and their expression did not interfere withthe expression of AAV2 Rep78/52 or with the rescue of rAAV2 genomereplication in the co-infected Sf9 cells. The replicative form (RF) anddouble RF of the rAAV2 DNA are indicated. (C) Vector purification.Infected Sf9 cells from 200 mL culture were used to purify the vector ona CsCl gradient. Fractions were collected and quantified for DRP.Purified vector was visualized under an electron microscope usingnegative staining. The pictograph reveals fully-packaged virions ofabout 25 nm in diameter. (D) Side-by-side comparison of rAAV2/HBoV1 andrAAV2/2 vector production in Sf9 cells (from 200 mL of Sf9-cellculture). The BEV used to generate rMV2 were Bac-(ITR)GFP andBac-Rep/(AAV2)Cap (kindly provided by the Kotin laboratory). Vectorswere purified using a CsCl gradient, and quantified as DRP/prep using aGFP probe. (E) Functionality of the rAAV2/HBoV1 vectors produced in Sf9cells was as active as that produced from 293 cells. Data represent theRLU in CuFi-ALI cultures apically infected with vector produced in Sf9or 293 cells. MOI of 10K were applied and cells were lysed forluciferase assays at 48 hours p.i.

FIG. 11. HBoV1 can encapsidate a recombinant parvoviral genome largerthan 5.5 Kb-packaging of a 5.9-kb AAV2 genome into the HBoV1 capsid.rAAV2/HBoV1 vectors were produced from three transfer plasmids, eachwith a genome of a different size, as indicated. The vector yieldrepresents production from transfected 293 cells in eight 150-mm plates,following CsCl-gradient ultracentrifugation. Viral DNA was extracted,resolved on a 0.9% alkaline gel, and visualized using a ³²P-labeled CFTRprobe.

FIGS. 12A-12B. The rAAV2/HBoV1 vector efficiently transduces ciliatedand K18-positive epithelial cells in HAE-ALI cultures. The indicatedvector was applied apically at an MOI of 10 k; expression of the mCherryreporter protein (Red) identifies the transduced airway cells. At 10days p.i., the HAE was (A) fixed and stained with anti-β-tubulin IV(Green), a marker of ciliated cells, and (B) trypsinized, cytospun ontoa slide, fixed, and stained with anti-K18 (Green), for both ciliated andnon-ciliated columnar cells. Confocal images were taken at ×100. DAPI:nucleus.

FIGS. 13A-13C. rAAV2 genome constructs for CFTR gene delivery. (A)Screening for short synthetic enhancers for the tg83 synthetic promoter.HAE-ALI were infected with rAAV2/2 vectors carrying a tg83-drivenluciferase cassette and various enhancers. At 3 days p.i., the infectedHAE were analyzed for luciferease activity (RLU). (B) Correction (30%)of Cl⁻ transport in CF HAE-ALI by the rAAV2/HBoV1 vector is moreeffective than that achieved with rAAV2/2. CF HAE-ALI cultures were mockinfected or infected with the vector depicted (n=6 for each condition),from the apical side and at the indicated MOI. At 10 days p.i., theinfected CF HAE were evaluated for correction of the CF phenotype, basedon changes in transepithelial short circuit current (isc), using anepithelial voltage clamp and a self-contained Ussing chamber system. Atan MOI of 10 k, rAAV2/HBoV1 (AV2/HBc) restored about 30% ofCFTR-mediated transepithelial Cl⁻ transport as that of the normal HAE(n=13). rAAV2/2-CFTR vectors were inefficient at correcting the CFphenotype, even at an MOI of 50 k. (C) rAAV2 genome constructs forrAAV2/HBoV1 vector. rAAV2-CFTR genome constructs that include a ciliatedcell-specific promoter (FOXJ1) or synthetic promoter/enhancer (F5tg83),or incorporate post-transcriptional elements (miR) are shown, and willbe packaged into the HBoV1 capsid.

FIGS. 14A-14D. Schematic approach for correcting a defective CFTR mRNAusing a SMaRT vector. (A) The rAAV2 genome AV2.CMV-PTM24CF, which ispseudotyped in the HBoV1 capsid, and the effectiveness of SMaRT will betested by apical infection of the CF HAE. (B) Sequence of thetrans-splicing domain of AV2.CMV-PTM24CF, which consists of: the 133-ntPTM24 binding sequence (in blue, complementary to a 133-nt BD RNAsequence at intron 9) following with endogenous branch point (BP inred), polypyrimidine tract (PPT in green) and the 3′SS (CAG) (SEQ ID NO:46). (C) Schematic representation of structure of the CFTR pre-mRNA andtargeting mechanism. Some critical mutations that cause defects in, orthe lack of, CFTR protein, lie in and downstream of exon 10, asindicated. (D) The proposed new rAAV2 genome AV2.CBA-PTM24CF-3UTR.

FIG. 15. rAAV2/HBoV1 Transduction in New Born Ferret. 3-day old ferretpup was infected with 4×10¹⁰ DRP of AAV2/HBoV1.F5tg83luc throughintratracheal injection. The volume of the inoculum is 300 μL withdoxorubicin at the final concentration of 250 μM. The animal wassacrificed 1 week post-infection, the airway cassette was harvested anddissected. 200 μL reporter lysis buffer (for each piece of tissue) wasused to extracted the protein from the trachea and the lobes of the lung(six lobes varied in size and weight). Luciferase activity (RLU) wasmeasured from the protein extraction and normalized to per mg of thetissue (wet weight). 100 ng genome DNA of each tissue sample was usedfor probing the amount of vector genome copies (VGC) by TaqMan PCR.Uninfected lungs from a ferret pup are shown as a negative control.

FIGS. 16A-16G. Exemplary swine, feline and canine bocavirus genome andVP sequences (SEQ ID NOs: 37-45).

DETAILED DESCRIPTION Definitions

A “vector” as used herein refers to a macromolecule or association ofmacromolecules that comprises or associates with a polynucleotide andwhich can be used to mediate delivery of the polynucleotide to a cell,either in vitro or in vivo. Illustrative vectors include, for example,plasmids, viral vectors, liposomes and other gene delivery vehicles. Thepolynucleotide to be delivered, sometimes referred to as a “targetpolynucleotide” or “transgene,” may comprise a coding sequence ofinterest in gene therapy (such as a gene encoding a protein oftherapeutic or interest), a coding sequence of interest in vaccinedevelopment (such as a polynucleotide expressing a protein, polypeptideor peptide suitable for eliciting an immune response in a mammal),and/or a selectable or detectable marker.

“AAV” is adeno-associated virus, and may be used to refer to thenaturally occurring wild-type virus itself or derivatives thereof. Theterm covers all subtypes, serotypes and pseudotypes, and both naturallyoccurring and recombinant forms, except where required otherwise. Asused herein, the term “serotype” refers to an AAV which is identified byand distinguished from other AAVs based on capsid protein reactivitywith defined antisera, e.g., there are eight serotypes of primate AAVs,AAV-1 to MV-8. For example, serotype AAV2 is used to refer to an MVwhich contains capsid proteins encoded from the cap gene of MV 2 and agenome containing 5′ and 3′ ITR sequences from the same AAV2 serotype.For each example illustrated herein the description of the vector designand production describes the serotype of the capsid and 5′-3′ ITRsequences. The abbreviation “rAAV” refers to recombinantadeno-associated virus, also referred to as a recombinant AAV vector (or“rAAV vector”).

BoV is bocavirus, and may be used to refer to the naturally occurringwild-type virus itself or derivatives thereof. The term covers allsubtypes, serotypes and pseudotypes, and both naturally occurring andrecombinant forms, except where required otherwise. As used herein, theterm “serotype” refers to a BoV, which is identified by anddistinguished from other BoVs based on capsid protein reactivity withdefined antisera, e.g., there are four known serotypes of humanbocavirus (HBoV), HBoV1, HBoV2, HBoV3, and HBoV4. However, included inBoV are serotypes derived from other non-human mammals such as swineBoV. Like for MV, different serotypes of HBoV and BoV can have differenttropisms that infect different cell types and organs.

rAAV/HBoV is a chimeric vector which is composed of HBoV capsids and arAAV genome. In such a chimeric virus there is no genetic informationfrom HBoV within the genome. The rAAV genome may be from any serotype ofMV.

rAAV/BoV is a chimeric vector which is composed of a non-human BoVcapsids and a rAAV genome. In such a chimeric virus there is no geneticinformation from BoV within the genome. The rAAV genome may be from anyserotype of AAV.

Tropism as used herein, is a term referring to the ability of aparticular viral serotype to productively infect cells of differingphenotypes or organs to deliver their genomic information to thenucleus.

“Transduction” or “transducing” as used herein, are terms referring to aprocess for the introduction of an exogenous polynucleotide, e.g., atransgene in rAAV vector, into a host cell leading to expression of thepolynucleotide, e.g., the transgene in the cell. The process includesone or more of 1) endocytosis of the chimeric virus, 2) escape fromendosomes or other intracellular compartments in the cytosol of a cell,3) trafficking of the viral particle or viral genome to the nucleus, 4)uncoating of the virus particles, and generation of expressible doublestranded MV genome forms, including circular intermediates. The rAAVexpressible double stranded form may persist as a nuclear episome oroptionally may integrate into the host genome. The alteration of any ora combination of endocytosis of the chimeric virus after it has bound toa cell surface receptor, escape from endosomes or other intracellularcompartments to the cytosol of a cell, trafficking of the viral particleor viral genome to the nucleus, or uncoating of the virus particles, andgeneration of expressive double stranded AAV genome forms, includingcircular intermediates, by an agent of the invention, e.g., a proteasomeinhibitor, may result in altered expression levels or persistence ofexpression, or altered trafficking to the nucleus, or altered types orrelative numbers of host cells or a population of cells expressing theintroduced polynucleotide. Altered expression or persistence of apolynucleotide introduced via the chimeric virus can be determined bymethods well known to the art including, but not limited to, proteinexpression, e.g., by ELISA, flow cytometry and Western blot, measurementof and DNA and RNA production by hybridization assays, e.g., Northernblots, Southern blots and gel shift mobility assays. The agents of theinvention may alter, enhance or increase viral endocytosis, escape fromendosomes or other intracellular cytosolic compartments, and traffickinginto or to the nucleus, uncoating of the viral particles in the nucleus,and/or increasing concatamerization or generation of double strandedexpressible forms of the rAAV genome in the nucleus, so as to alterexpression of the introduced polynucleotide, e.g., a transgene in a rAAVvector, in vitro or in vivo. Methods used for the introduction of theexogenous polynucleotide include well-known techniques such astransfection, lipofection, viral infection, transformation, andelectroporation, as well as non-viral gene delivery techniques. Theintroduced polynucleotide may be stably or transiently maintained in thehost cell.

“Increased transduction or transduction frequency”, “alteredtransduction or transduction frequency”, or “enhanced transduction ortransduction frequency” refers to an increase in one or more of theactivities described above in a treated cell relative to an untreatedcell. Agents of the invention which increase transduction efficiency maybe determined by measuring the effect on one or more transductionactivities, which may include measuring the expression of the transgene,measuring the function of the transgene, or determining the number ofparticles necessary to yield the same transgene effect compared to hostcells not treated with the agents.

“Proteasome modulator” refers to an agent or class of agents which alteror enhance rAAV including chimeric virus transduction or transductionfrequencies by interacting with, binding to, or altering the functionof, and/or trafficking or location of the proteasome. Proteasomemodulators may have other cellular functions as described in the art,e.g., such as doxyrubicin, an antibiotic. Proteasome modulators includeproteasome inhibitors, e.g., such as tripeptidyl aldehydes (MG132, i.e.,Z-LLL or MG101, i.e., LLnL), bortezomib (Velcade), agents that inhibitcalpains, cathepsins, cysteine proteases, and/or chymotrypsin-likeprotease activity of proteasomes (Wagner et al., 2002; Young et al.,2000; Seisenberger et al., 2001).

“Gene delivery” refers to the introduction of an exogenouspolynucleotide into a cell for gene transfer, and may encompasstargeting, binding, uptake, transport, localization, repliconintegration and expression.

“Gene transfer” refers to the introduction of an exogenouspolynucleotide into a cell which may encompass targeting, binding,uptake, transport, localization and replicon integration, but isdistinct from and does not imply subsequent expression of the gene.

“Gene expression” or “expression” refers to the process of genetranscription, translation, and post-translational modification.

A “detectable marker gene” is a gene that allows cells carrying the geneto be specifically detected (e.g., distinguished from cells which do notcarry the marker gene). A large variety of such marker genes are knownin the art.

A “selectable marker gene” is a gene that allows cells carrying the geneto be specifically selected for or against, in the presence of acorresponding selective agent. By way of illustration, an antibioticresistance gene can be used as a positive selectable marker gene thatallows a host cell to be positively selected for in the presence of thecorresponding antibiotic. A variety of positive and negative selectablemarkers are known in the art, some of which are described below.

An “rAAV vector” as used herein refers to an AAV vector comprising apolynucleotide sequence not of AAV origin (i.e., a polynucleotideheterologous to AAV), typically a sequence of interest for the genetictransformation of a cell. In preferred vector constructs of thisinvention, the heterologous polynucleotide is flanked by one or two MVinverted terminal repeat sequences (1TRs). The term rAAV vectorencompasses both rAAV vector particles and rAAV vector plasmids.

A “Chimeric virus” or “Chimeric viral particle” refers to a viralparticle composed of at least one capsid protein and an encapsidatedpolynucleotide, which is from a different virus.

A “helper virus” for MV refers to a virus that allows AAV (e.g.,wild-type AAV) to be replicated and packaged by a mammalian cell. Avariety of such helper viruses for AAV are known in the art, includingadenoviruses, herpes viruses and poxviruses such as vaccinia. Theadenoviruses encompass a number of different subgroups, althoughAdenovirus type 5 of subgroup C is most commonly used. Numerousadenoviruses of human, non-human mammalian and avian origin are knownand available from depositories such as the ATCC.

An “infectious” virus or viral particle is one that comprises apolynucleotide component, which it is capable of delivering into a cellfor which the viral species is trophic. The term does not necessarilyimply any replication capacity of the virus.

The term “polynucleotide” refers to a polymeric form of nucleotides ofany length, including deoxyribonucleotides or ribonucleotides, oranalogs thereof. A polynucleotide may comprise modified nucleotides,such as methylated or capped nucleotides and nucleotide analogs, and maybe interrupted by non-nucleotide components. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The term polynucleotide, as used herein, refersinterchangeably to double- and single-stranded molecules. Unlessotherwise specified or required, any embodiment of the inventiondescribed herein that is a polynucleotide encompasses both thedouble-stranded form and each of two complementary single-stranded formsknown or predicted to make up the double-stranded form.

A “transcriptional regulatory sequence” or “TRS,” as used herein, refersto a genomic region that controls the transcription of a gene or codingsequence to which it is operably linked. Transcriptional regulatorysequences of use in the present invention generally include at least onetranscriptional promoter and may also include one or more enhancersand/or terminators of transcription.

“Operably linked” refers to an arrangement of two or more components,wherein the components so described are in a relationship permittingthem to function in a coordinated manner. By way of illustration, atranscriptional regulatory sequence or a promoter is operably linked toa coding sequence if the TRS or promoter promotes transcription of thecoding sequence. An operably linked TRS is generally joined in cis withthe coding sequence, but it is not necessarily directly adjacent to it.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is compared. For example, apolynucleotide introduced by genetic engineering techniques into adifferent cell type is a heterologous polynucleotide (and, whenexpressed, can encode a heterologous polypeptide). Similarly, a TRS orpromoter that is removed from its native coding sequence and operablylinked to a different coding sequence is a heterologous TRS or promoter.

“Packaging” as used herein refers to a series of subcellular events thatresults in the assembly and encapsidation of a viral vector. Thus, whena suitable vector is introduced into a packaging cell line underappropriate conditions, it can be assembled into a viral particle.Functions associated with packaging of viral vectors are describedherein and in the art.

A “terminator” refers to a polynucleotide sequence that tends todiminish or prevent read-through transcription (i.e., it diminishes orprevent transcription originating on one side of the terminator fromcontinuing through to the other side of the terminator). The degree towhich transcription is disrupted is typically a function of the basesequence and/or the length of the terminator sequence. In particular, asis well known in numerous molecular biological systems, particular DNAsequences, generally referred to as “transcriptional terminationsequences,” are specific sequences that tend to disrupt read-throughtranscription by RNA polymerase, presumably by causing the RNApolymerase molecule to stop and/or disengage from the DNA beingtranscribed. Typical examples of such sequence-specific terminatorsinclude polyadenylation (“polyA”) sequences, e.g., SV40 polyA. Inaddition to or in place of such sequence-specific terminators,insertions of relatively long DNA sequences between a promoter and acoding region also tend to disrupt transcription of the coding region,generally in proportion to the length of the intervening sequence. Thiseffect presumably arises because there is always some tendency for anRNA polymerase molecule to become disengaged from the DNA beingtranscribed, and increasing the length of the sequence to be traversedbefore reaching the coding region would generally increase thelikelihood that disengagement would occur before transcription of thecoding region was completed or possibly even initiated. Terminators maythus prevent transcription from only one direction (“uni-directional”terminators) or from both directions (“bi-directional” terminators), andmay be comprised of sequence-specific termination sequences orsequence-non-specific terminators or both. A variety of such terminatorsequences are known in the art; and illustrative uses of such sequenceswithin the context of the present invention are provided below.

“Host cells,” “cell lines,” “cell cultures,” “packaging cell line” andother such terms denote higher eukaryotic cells, e.g., mammalian cells,such human cells, useful in the present invention. These cells can beused as recipients for recombinant vectors, viruses or other transferpolynucleotides, and include the progeny of the original cell that wastransduced. It is understood that the progeny of a single cell may notnecessarily be completely identical (in morphology or in genomiccomplement) to the original parent cell.

A “therapeutic gene,” “prophylactic gene,” “target polynucleotide,”“transgene,” “gene of interest” and the like generally refer to a geneor genes to be transferred using a vector. Typically, in the context ofthe present invention, such genes are located within the rAAV vector(which vector is flanked by inverted terminal repeat (ITR) regions andthus can be replicated and encapsidated into rAAV particles). Targetpolynucleotides can be used in this invention to generate rAAV vectorsfor a number of different applications. Such polynucleotides include,but are not limited to: (i) polynucleotides encoding proteins useful inother forms of gene therapy to relieve deficiencies caused by missing,defective or sub-optimal levels of a structural protein or enzyme; (ii)polynucleotides that are transcribed into anti-sense molecules; (iii)polynucleotides that are transcribed into decoys that bind transcriptionor translation factors; (iv) polynucleotides that encode cellularmodulators such as cytokines; (v) polynucleotides that can makerecipient cells susceptible to specific drugs, such as the herpes virusthymidine kinase gene; and (vi) polynucleotides for cancer therapy, suchas E1A tumor suppressor genes or p53 tumor suppressor genes for thetreatment of various cancers. To effect expression of the transgene in arecipient host cell, it is operably linked to a promoter, either its ownor a heterologous promoter. A large number of suitable promoters areknown in the art, the choice of which depends on the desired level ofexpression of the target polynucleotide; whether one wants constitutiveexpression, inducible expression, cell-specific or tissue-specificexpression, etc. The rAAV vector may also contain a selectable marker.

A “gene” refers to a polynucleotide containing at least one open readingframe that is capable of encoding a particular protein after beingtranscribed and translated.

“Recombinant,” as applied to a polynucleotide means that thepolynucleotide is the product of various combinations of cloning,restriction and/or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature. Arecombinant virus is a viral particle comprising a recombinantpolynucleotide. The terms respectively include replicates of theoriginal polynucleotide construct and progeny of the original virusconstruct.

A “control element” or “control sequence” is a nucleotide sequenceinvolved in an interaction of molecules that contributes to thefunctional regulation of a polynucleotide, including replication,duplication, transcription, splicing, translation, or degradation of thepolynucleotide. The regulation may affect the frequency, speed, orspecificity of the process, and may be enhancing or inhibitory innature. Control elements known in the art include, for example,transcriptional regulatory sequences such as promoters and enhancers. Apromoter is a DNA region capable under certain conditions of binding RNApolymerase and initiating transcription of a coding region usuallylocated downstream (in the 3′ direction) from the promoter. Promotersinclude AAV promoters, e.g., P5, P19, P40 and AAV ITR promoters, as wellas heterologous promoters.

An “expression vector” is a vector comprising a region which encodes apolypeptide of interest, and is used for effecting the expression of theprotein in an intended target cell. An expression vector also comprisescontrol elements operatively linked to the encoding region to facilitateexpression of the protein in the target. The combination of controlelements and a gene or genes to which they are operably linked forexpression is sometimes referred to as an “expression cassette,” a largenumber of which are known and available in the art or can be readilyconstructed from components that are available in the art.

“Genetic alteration” refers to a process wherein a genetic element isintroduced into a cell other than by mitosis or meiosis. The element maybe heterologous to the cell, or it may be an additional copy or improvedversion of an element already present in the cell. Genetic alterationmay be effected, for example, by transfecting a cell with a recombinantplasmid or other polynucleotide through any process known in the art,such as electroporation, calcium phosphate precipitation, or contactingwith a polynucleotide-liposome complex. Genetic alteration may also beeffected, for example, by transduction or infection with a DNA or RNAvirus or viral vector. The genetic element may be introduced into achromosome or mini-chromosome in the cell; but any alteration thatchanges the phenotype and/or genotype of the cell and its progeny isincluded in this term.

A cell is said to be “stably” altered, transduced or transformed with agenetic sequence if the sequence is available to perform its functionduring extended culture of the cell in vitro. In some examples, such acell is “inheritably” altered in that a genetic alteration is introducedwhich is also inheritable by progeny of the altered cell.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to polymers of amino acids of any length. The terms also encompassan amino acid polymer that has been modified; for example, disulfidebond formation, glycosylation, acetylation, phosphorylation, lipidation,or conjugation with a labeling component. Polypeptides such as “CFTR”and the like, when discussed in the context of gene therapy andcompositions therefor, refer to the respective intact polypeptide, orany fragment or genetically engineered derivative thereof, that retainsthe desired biochemical function of the intact protein. Similarly,references to CFTR, and other such genes for use in gene therapy(typically referred to as “transgenes” to be delivered to a recipientcell), include polynucleotides encoding the intact polypeptide or anyfragment or genetically engineered derivative possessing the desiredbiochemical function.

An “isolated” plasmid, virus, or other substance refers to a preparationof the substance devoid of at least some of the other components thatmay also be present where the substance or a similar substance naturallyoccurs or is initially prepared from. Thus, for example, an isolatedsubstance may be prepared by using a purification technique to enrich itfrom a source mixture. Enrichment can be measured on an absolute basis,such as weight per volume of solution, or it can be measured in relationto a second, potentially interfering substance present in the sourcemixture.

A preparation of AAV is said to be “substantially free” of helper virusif the ratio of infectious AAV particles to infectious helper virusparticles is at least about 10²:1; e.g., at least about 10⁴:1, includingat least about 10⁶:1 or at least about 10⁸:1. Preparations may also befree of equivalent amounts of helper virus proteins (i.e., proteins aswould be present as a result of such a level of helper virus if thehelper virus particle impurities noted above were present in disruptedform). Viral and/or cellular protein contamination can generally beobserved as the presence of Coomassie staining bands on SDS gels (e.g.,the appearance of bands other than those corresponding to the AAV capsidproteins VP1, VP2 and VP3).

“Efficiency” when used in describing viral production, replication orpackaging refers to useful properties of the method: in particular, thegrowth rate and the number of virus particles produced per cell. “Highefficiency” production indicates production of at least 100 viralparticles per cell; e.g., at least about 10,000 or at least about100,000 particles per cell, over the course of the culture periodspecified.

An “individual” or “subject” treated in accordance with this inventionrefers to vertebrates, particularly members of a mammalian species, andincludes but is not limited to domestic animals, sports animals, andprimates, including humans.

“Treatment” of an individual or a cell is any type of intervention in anattempt to alter the natural course of the individual or cell at thetime the treatment is initiated, e.g., eliciting a prophylactic,curative or other beneficial effect in the individual. For example,treatment of an individual may be undertaken to decrease or limit thepathology caused by any pathological condition, including (but notlimited to) an inherited or induced genetic deficiency, infection by aviral, bacterial, or parasitic organism, a neoplastic or aplasticcondition, or an immune system dysfunction such as autoimmunity orimmunosuppression. Treatment includes (but is not limited to)administration of a composition, such as a pharmaceutical composition,and administration of compatible cells that have been treated with acomposition. Treatment may be performed either prophylactically ortherapeutically; that is, either prior or subsequent to the initiationof a pathologic event or contact with an etiologic agent.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, virology,microbiology, recombinant DNA, and immunology, which are within theskill of the art. Such techniques are explained fully in the literature.See e.g., Sambrook et al., 1989; Gait, 1984; Freshney, 1987; the seriesMethods in Enzymology (Academic Press, Inc.); Miller et al., 1987; Weiret al., 1996; Ausubel et al., 1998; Coligan et al., 1991; Coligan etal., 1995; and Scopes 1994.

I. Chimeric Viruses

Human airway epithelial cells are highly resistant to infection by mostviral vectors included the adeno-associated virus (rAAV), the mostwidely used gene therapy vector in clinical trials. Human Bocavirus 1(HBoV1), an autonomous human parvovirus which is likely an etiologicalagent of acute respiratory tract infections (ARTI) associated withwheezing in infants and young children (Allander et al., 2007;Christensen et al., 2010; Deng et al., 2012; Don et al., 2010),efficiently infects HAE from the apical membrane, resulting inreplication of progeny viruses and cytopathology (Huang et al., 2012a).Impressively, HBoV1 infection of HAE at extremely low multiplicities ofinfection (MOI) of 10⁻³ DNase-resistant particles (DRP) per cell resultsin a productive infection (see Example 2). Recently, the full-length5543-nt HBoV1 complete genome (including terminal palindromic sequencesat both ends) was cloned, and cell culture systems for HBoV1 productionhave been established (Example 1). Given the high efficiency of HBoV1infection from the apical surface of HAE, HoBV1 was hypothesized to besuitable for engineering recombinant vectors for human airway genetherapy.

HBoV1 is a relative of AAV and other Parvoviridae family members. HBoV1belongs to the genus Bocavirus, while AAV is in the genus Dependovirus(Tijssen et al., 2011). HBoV1 and AAV are both small single-stranded DNAviruses, but 90% of encapsidated HBoV1 genomes are of the minus strand,while for AAV, an equal ratio of plus and minus strands are encapsidated(Schildgen et al., 2012). These two viruses differ greatly in theirlytic phase life cycle; MV requires co-infection with a helper virus,while HBoV1 autonomously replicates progeny in permissive cells (Huanget al., 2012a; Dijkman et al., 2009). The HBoV1 genome size is 5543 nt,18.5% (863 nt) larger than that of AAV2 (4679-nt), and its structuralfeatures include asymmetrical hairpins with unique palindromic sequencesat 5′ (140 nt) and 3′ (200 nt) termini, which are involved inreplication and encapsidation, and a single P5 promoter that transcribesall viral structural and non-structural proteins (Huang et al., 2012;Chen et al., 2010). This is in contrast to the inverted terminal repeatsand multiple internal promoters found in AAV genomes. The HBoV1 genomeencodes three major open reading frames (ORF). Two of them code fornonstructural proteins, NS1/NS2 and NP1, which are essential for virusreplication. The third ORF encodes two structural capsid proteins VP1and VP2. By contrast, the MV cap ORF encodes three capsid proteins, VP1,VP2, and VP3 (Schidgen et al., 2012). HBoV1 capsid surface topologypossesses common features with other parvoviruses (icosahedral capsid),and is most closely similar to human parvovirus B19 (Gurda et al.,2010). Like the cloned AAV genome, a plasmid that encodes the HBoV1proviral genome is infectious and can be used to produce infectiousparticles through transfection into HEK 293 cells without the need forhelper virus co-infection (Example 1).

Cross-genera pseudopackaging between Parvoviridae was first establishedwhen a rAAV genome was encapsidated into a human parvovirus B19 capsid(Ponnazhagan et al., 1998). This resultant cross-genera chimera was ableto deliver the rAAV genome into human bone marrow cells that areresistant to rAAV infection (Ponnazhagan et al., 1998). Thus, it washypothesized that pseudotyping the rAAV genome into HBoV1 capsid mightcreate a novel chimeric vector with unique properties for gene therapyof CF and other pulmonary diseases.

The production of rHBoV1 vectors and chimeric rAAV2/HBoV1 vectors isdescribed herein below. The first virus was a conventional recombinantvector (a rHBoV1 vector). An open reading frame disrupted or gutted HBoVgenome carrying a foreign gene is packaged inside the HBoV1 capsid.rHBoV1 vector is produced in HEK293 cells by trans-complementation fromthe co-transfection of rHBoV1 proviral plasmid and HBoV1 helper plasmid.The rHBoV1 proviral plasmid harbors a foreign gene (of about 5.2 kb inlength or more, which can accommodate a heterologous promoter, e.g., astrong promoter, operably linked to an open reading frame for theforeign gene) and all the cis-elements for replication and package, thehelper plasmid encodes only the expression cassette for HBoV viralproteins. One important feature of the HBoV1 virus is that its genomeautonomously replicates in permissive cells, in contrast to rAAV, whichis a dependent parvovirus and needs helper virus coinfection forreplication.

With the success in trans-complementation for rHBoV1 vector production,a so-called replicative rHBoV1 vector was developed by retaining thecoding sequences for HBoV1 rep genes but replacing the structural geneby a transgene. This type of vector can deliver a high level oftherapeutic gene expression in the airway cells for the therapy such asCF, AAT deficiency, COPD, or lung cancers. Such a replicating HBoV1vector could have high utility as a vaccine against WT HBoV1 infections.

Another vector developed was an AAV2-HBoV1 chimeric virus, whichpackages a rAAV genome into a HBoV1 capsid particle. The vector was alsoproduced in HEK293 cells with a procedure similar for rAAV vector, butthe capsid genes are substituted by HBoV1 capsids. This AAV/HBoV1 vectorcombines both the advantages of MV and HBoV1 transduction biology, withless safety concerns than the rHBoV1 vector since rAAV vector genomeshave been extensively studied in many pre-clinical research and clinicaltrials, but higher airway cell tropism than rAAV. More importantly, thelarge HBoV1 package capacity makes it possible to encapsidate anoversized rAAV genome up to about 5.5 kb or about 6.0 kb. The 20%greater capacity than rAAV is enough to house a strong expressioncassette for effective gene expression. A rAAV genome providesadvantages of persistent gene expression by the stable circulartransduction intermediates and double stranded genome concatemers.Indeed, AAV/HBoV1 vectors featured more persistent transgene expressionthan the rHBoV1 vector. Furthermore, the rescue and replication of rAAVgenomes in HEK293 cells was very efficient, so that the production yieldof the AAV/HBoV1 vector was also better than an rHBoV1 vector.

Utilizing the larger packaging capacity of HBoV1, a rAAV2/1-1BoV1-CFTRvector was prepared that harbors a 5.5 kb oversized rAAV genome with a5.2 kb CFTR expression cassette having a strong chimeric promoter thatincluded the human CMV immediate gene enhancer and the chicken β-actinpromoter (CBA promoter). That vector demonstrated about 30% restorationof CFTR-mediate chloride currents in CF HAE following apical infection.Therefore, the vector can efficiently deliver normal CFTR proteinexpression on the surface of the airway epithelial cells and correct thedefective CFTR specific chloride transport in the CF HAE. In addition,the HBoV1 genome can encapsidate the self-complementary double strandedform of a rAAV genome of about 2.7 kb to about 2.8 kb in length, whichvector can bypass genome conversion and allow for enhanced or more rapidtransgene expression. The AAV/HBoV chimeric vectors could also beexpanded to other therapies for other lung diseases such asalpha-antitrypsin deficiency, asthma, and lung cancer, as well asvaccination against wild-type HBoV infections in infants.

The capsids and/or genomes of the viruses of the invention may bechimeric, e.g., as a result of directed evolution (see, e.g., Li et al.,2009).

II. rAAV Vectors

Besides prophylactic or therapeutic gene products, recombinant AAVvectors and/or viruses can also comprise polynucleotides that do notencode proteins, including, e.g., polynucleotides encoding for antisensemRNA (the complement of mRNA) which can be used to block the translationof normal mRNA by forming a duplex with it, and polynucleotides thatencode ribozymes (RNA catalysts). In addition selected pairs of rAAVvectors having portions of open reading frames flanked by appropriatelyplaced splice acceptor sites and/or splice donor sites, or havingtranscription regulatory sequences such as a heterologous enhancer, aheterologous promoter, or a heterologous enhancer and a promoter, may beemployed. See, e.g., U.S. Pat. No. 6,436,392, the disclosure of which isincorporated by reference herein. For example, a first MV vector mayinclude a first DNA segment comprising a 5′-inverted terminal repeat ofMV: a second DNA segment comprising a promoter operably linked to a DNAfragment comprising an exon of a gene and a splice donor site, whereinthe second DNA segment does not encode a full-length polypeptide; and athird DNA segment comprising a 3′-inverted terminal repeat of MV: and asecond AAV vector comprising linked: a first DNA segment comprising a5′-inverted terminal repeat of AAV; a second DNA segment comprising asplice acceptor site and a DNA fragment with at least one other exonwhich together with the DNA segment of the first MV vector encodes afull-length polypeptide; and a third DNA segment comprising a3′-inverted terminal repeat of AAV. In one example, a first MV vectorincludes the following:a first nucleic acid segment comprising a5′-inverted terminal repeat of MV; a second nucleic acid segmentcomprising a portion of a gene which includes a transcriptionalregulatory region; a third nucleic acid segment comprising a splicedonor site; and a fourth nucleic acid segment comprising a 3′-invertedterminal repeat of MV; and a second AAV vector comprising linked: afirst nucleic acid segment comprising a 5′-inverted terminal repeat ofMV; a second nucleic acid segment comprising a splice acceptor site; athird nucleic acid segment comprising a portion of a gene which togetherwith the nucleic acid segment of the first MV vector comprises a genecomprising an open reading frame which encodes a functional polypeptide;and a fourth nucleic acid segment comprising a 3′-inverted terminalrepeat of MV. In a further example, a first AAV vector includes thefollowing: a first nucleic acid segment comprising a 5′-invertedterminal repeat of AAV; a second nucleic acid segment comprising asplice acceptor site; a third nucleic acid segment comprising a portionof a gene; and a fourth nucleic acid segment comprising a 3′-invertedterminal repeat of MV; and a second composition comprising a second AAVvector comprising: a first nucleic acid segment comprising a 5′-invertedterminal repeat of AAV; a second nucleic acid segment comprising aportion of a gene which together with the nucleic acid segment abovehaving the portion comprises a gene comprising an open reading framewhich encodes a functional polypeptide, wherein the portion of the geneincludes a transcriptional regulatory region; a third nucleic acidsegment comprising a splice donor site; a fourth nucleic acid segmentcomprising a 3′-inverted terminal repeat of MV; which vectors in a hostcell yield a RNA transcript which comprises sequences from the first MVvector linked to sequences from the second AAV vector, which sequencesare positioned so that the splice donor site is 5′ to the spliceacceptor site, and which transcript is spliced to a mRNA which encodesthe functional protein.

Adeno-associated viruses of any serotype are suitable to prepare rAAV,since the various serotypes are functionally and structurally related,even at the genetic level (see, e.g., Blacklow, 1988; and Rose, 1974).All AAV serotypes apparently exhibit similar replication propertiesmediated by homologous rep genes; and all generally bear three relatedcapsid proteins such as those expressed in AAV2. The degree ofrelatedness is further suggested by heteroduplex analysis which revealsextensive cross-hybridization between serotypes along the length of thegenome; and the presence of analogous self-annealing segments at thetermini that correspond to ITRs. The similar infectivity patterns alsosuggest that the replication functions in each serotype are undersimilar regulatory control. Among the various MV serotypes, AAV2 is mostcommonly employed.

An AAV vector of the invention typically comprises a polynucleotide thatis heterologous to MV. The polynucleotide is typically of interestbecause of a capacity to provide a function to a target cell in thecontext of gene therapy, such as up- or down-regulation of theexpression of a certain phenotype. Such a heterologous polynucleotide or“transgene,” generally is of sufficient length to provide the desiredfunction or encoding sequence.

Where transcription of the heterologous polynucleotide is desired in theintended target cell, it can be operably linked to its own or to aheterologous promoter, depending for example on the desired level and/orspecificity of transcription within the target cell, as is known in theart. Various types of promoters and enhancers are suitable for use inthis context. Constitutive promoters provide an ongoing level of genetranscription, and may be preferred when it is desired that thetherapeutic or prophylactic polynucleotide be expressed on an ongoingbasis. Inducible promoters generally exhibit low activity in the absenceof the inducer, and are up-regulated in the presence of the inducer.They may be preferred when expression is desired only at certain timesor at certain locations, or when it is desirable to titrate the level ofexpression using an inducing agent. Promoters and enhancers may also betissue-specific: that is, they exhibit their activity only in certaincell types, presumably due to gene regulatory elements found uniquely inthose cells.

Illustrative examples of promoters are the SV40 late promoter fromsimian virus 40, the Baculovirus polyhedron enhancer/promoter element,Herpes Simplex Virus thymidine kinase (HSV tk), the immediate earlypromoter from cytomegalovirus (CMV) and various retroviral promotersincluding LTR elements. Inducible promoters include heavy metal ioninducible promoters (such as the mouse mammary tumor virus (mMTV)promoter or various growth hormone promoters), and the promoters from T7phage which are active in the presence of T7 RNA polymerase. By way ofillustration, examples of tissue-specific promoters include varioussurfactin promoters (for expression in the lung), myosin promoters (forexpression in muscle), and albumin promoters (for expression in theliver). A large variety of other promoters are known and generallyavailable in the art, and the sequences of many such promoters areavailable in sequence databases such as the GenBank database.

Where translation is also desired in the intended target cell, theheterologous polynucleotide will preferably also comprise controlelements that facilitate translation (such as a ribosome binding site or“RBS” and a polyadenylation signal). Accordingly, the heterologouspolynucleotide generally comprises at least one coding regionoperatively linked to a suitable promoter, and may also comprise, forexample, an operatively linked enhancer, ribosome binding site andpoly-A signal. The heterologous polynucleotide may comprise one encodingregion, or more than one encoding regions under the control of the sameor different promoters. The entire unit, containing a combination ofcontrol elements and encoding region, is often referred to as anexpression cassette.

The heterologous polynucleotide is integrated by recombinant techniquesinto or in place of the AAV genomic coding region (i.e., in place of theAAV rep and cap genes), but is generally flanked on either side by AAVinverted terminal repeat (ITR) regions. This means that an ITR appearsboth upstream and downstream from the coding sequence, either in directjuxtaposition, e.g., (although not necessarily) without any interveningsequence of AAV origin in order to reduce the likelihood ofrecombination that might regenerate a replication-competent AAV genome.However, a single ITR may be sufficient to carry out the functionsnormally associated with configurations comprising two ITRs (see, forexample, WO 94/13788), and vector constructs with only one ITR can thusbe employed in conjunction with the packaging and production methods ofthe present invention.

The native promoters for rep are self-regulating, and can limit theamount of AAV particles produced. The rep gene can also be operablylinked to a heterologous promoter, whether rep is provided as part ofthe vector construct, or separately. Any heterologous promoter that isnot strongly down-regulated by rep gene expression is suitable; butinducible promoters may be preferred because constitutive expression ofthe rep gene can have a negative impact on the host cell. A largevariety of inducible promoters are known in the art; including, by wayof illustration, heavy metal ion inducible promoters (such asmetallothionein promoters); steroid hormone inducible promoters (such asthe MMTV promoter or growth hormone promoters); and promoters such asthose from 17 phage which are active in the presence of T7 RNApolymerase. One sub-class of inducible promoters are those that areinduced by the helper virus that is used to complement the replicationand packaging of the rAAV vector. A number of helper-virus-induciblepromoters have also been described, including the adenovirus early genepromoter which is inducible by adenovirus EIA protein; the adenovirusmajor late promoter; the herpesvirus promoter which is inducible byherpesvirus proteins such as VP16 or 1CP4; as well as vaccinia orpoxvirus inducible promoters.

Methods for identifying and testing helper-virus-inducible promotershave been described (see, e.g., WO 96/17947). Thus, methods are known inthe art to determine whether or not candidate promoters arehelper-virus-inducible, and whether or not they will be useful in thegeneration of high efficiency packaging cells. Briefly, one such methodinvolves replacing the p5 promoter of the MV rep gene with the putativehelper-virus-inducible promoter (either known in the art or identifiedusing well-known techniques such as linkage to promoter-less “reporter”genes). The AAV rep-cap genes (with p5 replaced), e.g., linked to apositive selectable marker such as an antibiotic resistance gene, arethen stably integrated into a suitable host cell (such as the HeLa orA549 cells exemplified below). Cells that are able to grow relativelywell under selection conditions (e.g., in the presence of theantibiotic) are then tested for their ability to express the rep and capgenes upon addition of a helper virus. As an initial test for rep and/orcap expression, cells can be readily screened using immunofluorescenceto detect Rep and/or Cap proteins. Confirmation of packagingcapabilities and efficiencies can then be determined by functional testsfor replication and packaging of incoming rAAV vectors. Using thismethodology, a helper-virus-inducible promoter derived from the mousemetallothionein gene has been identified as a suitable replacement forthe p5 promoter, and used for producing high titers of rAAV particles(as described in WO 96/17947).

Removal of one or more MV genes is in any case desirable, to reduce thelikelihood of generating replication-competent AAV (“RCA”). Accordingly,encoding or promoter sequences for rep, cap, or both, may be removed,since the functions provided by these genes can be provided in trans.

The resultant vector is referred to as being “defective” in thesefunctions. In order to replicate and package the vector, the missingfunctions are complemented with a packaging gene, or a pluralitythereof, which together encode the necessary functions for the variousmissing rep and/or cap gene products. The packaging genes or genecassettes are in one embodiment not flanked by AAV ITRs and in oneembodiment do not share any substantial homology with the rAAV genome.Thus, in order to minimize homologous recombination during replicationbetween the vector sequence and separately provided packaging genes, itis desirable to avoid overlap of the two polynucleotide sequences. Thelevel of homology and corresponding frequency of recombination increasewith increasing length of homologous sequences and with their level ofshared identity. The level of homology that will pose a concern in agiven system can be determined theoretically and confirmedexperimentally, as is known in the art. Typically, however,recombination can be substantially reduced or eliminated if theoverlapping sequence is less than about a 25 nucleotide sequence if itis at least 80% identical over its entire length, or less than about a50 nucleotide sequence if it is at least 70% identical over its entirelength. Of course, even lower levels of homology are preferable sincethey will further reduce the likelihood of recombination. It appearsthat, even without any overlapping homology, there is some residualfrequency of generating RCA. Even further reductions in the frequency ofgenerating RCA (e.g., by nonhomologous recombination) can be obtained by“splitting” the replication and encapsidation functions of MV, asdescribed by Allen et al., WO 98/27204).

The rAAV vector construct, and the complementary packaging geneconstructs can be implemented in this invention in a number of differentforms. Viral particles, plasmids, and stably transformed host cells canall be used to introduce such constructs into the packaging cell, eithertransiently or stably.

In certain embodiments of this invention, the MV vector andcomplementary packaging gene(s), if any, are provided in the form ofbacterial plasmids, AAV particles, or any combination thereof. In otherembodiments, either the MV vector sequence, the packaging gene(s), orboth, are provided in the form of genetically altered (preferablyinheritably altered) eukaryotic cells. The development of host cellsinheritably altered to express the AAV vector sequence, AAV packaginggenes, or both, provides an established source of the material that isexpressed at a reliable level.

A variety of different genetically altered cells can thus be used in thecontext of this invention. By way of illustration, a mammalian host cellmay be used with at least one intact copy of a stably integrated rAAVvector. An AAV packaging plasmid comprising at least an AAV rep geneoperably linked to a promoter can be used to supply replicationfunctions (as described in U.S. Pat. No. 5,658,776). Alternatively, astable mammalian cell line with an AAV rep gene operably linked to apromoter can be used to supply replication functions (see, e.g., Trempeet al., WO 95/13392); Burstein et al. (WO 98/23018); and Johnson et al.(U.S. Pat. No. 5,656,785). The AAV cap gene, providing the encapsidationproteins as described above, can be provided together with an AAV repgene or separately (see, e.g., the above-referenced applications andpatents as well as Allen et al. (WO 98/27204). Other combinations arepossible and included within the scope of this invention.

III. Uses of Chimeric Virus or rBoV

The chimeric virus or rBoV can be used for administration to anindividual for purposes of gene therapy or vaccination. Suitablediseases for therapy include but are not limited to those induced byviral, bacterial, or parasitic infections, various malignancies andhyperproliferative conditions, autoimmune conditions, and congenitaldeficiencies.

Gene therapy can be conducted to enhance the level of expression of aparticular protein either within or secreted by the cell. Vectors ofthis invention may be used to genetically alter cells either for genemarking, replacement of a missing or defective gene, or insertion of atherapeutic gene. Alternatively, a polynucleotide may be provided to thecell that decreases the level of expression. This may be used for thesuppression of an undesirable phenotype, such as the product of a geneamplified or overexpressed during the course of a malignancy, or a geneintroduced or overexpressed during the course of a microbial infection.Expression levels may be decreased by supplying a therapeutic orprophylactic polynucleotide comprising a sequence capable, for example,of forming a stable hybrid with either the target gene or RNA transcript(antisense therapy), capable of acting as a ribozyme to cleave therelevant mRNA or capable of acting as a decoy for a product of thetarget gene.

Vaccination can be conducted to protect cells from infection byinfectious pathogens. As the traditional vaccine methods, vectors ofthis invention may be used to deliver transgenes encoding viral,bacterial, tumor or fungal antigen and their subsequent expression inhost cells. The antigens, which expose to the immune system to evoke animmune response, can be in the form of virus-like particle vaccines orsubunit vaccines of virus-coding proteins. Alternatively, as the methodof passive immunolization, vectors of this invention might be used todeliver genes encoding neutralizing antibodies and their subsequentexpression in host non-hematopoietic tissues. The vaccine-likeprotection against pathogen infection can be conducted through directprovision of neutralizing antibody from vector-mediated transgeneexpression, bypassing the reliance on the natural immune system formounting desired humoral immune responses.

The introduction of the chimeric or rBoV vectors by the methods of thepresent invention may involve use of any number of delivery techniques(both surgical and non-surgical) which are available and well known inthe art. Such delivery techniques, for example, include vascularcatheterization, cannulization, injection, inhalation, endotracheal,subcutaneous, inunction, topical, oral, percutaneous, intra-arterial,intravenous, and/or intraperitoneal administrations. Vectors can also beintroduced by way of bioprostheses, including, by way of illustration,vascular grafts (PTFE and dacron), heart valves, intravascular stents,intravascular paving as well as other non-vascular prostheses. Generaltechniques regarding delivery, frequency, composition and dosage rangesof vector solutions are within the skill of the art.

In particular, for delivery of a vector of the invention to a tissue,any physical or biological method that will introduce the vector to ahost animal can be employed. Vector means both a bare recombinant vectorand vector DNA packaged into viral coat proteins, as is well known foradministration. Simply dissolving a chimeric or rHBoV vector inphosphate buffered saline has been demonstrated to be sufficient toprovide a vehicle useful for muscle tissue expression, and there are noknown restrictions on the carriers or other components that can becoadministered with the vector (although compositions that degrade DNAshould be avoided in the normal manner with vectors). Pharmaceuticalcompositions can be prepared as injectable formulations or as topicalformulations to be delivered to the muscles by transdermal transport.Numerous formulations for both intramuscular injection and transdermaltransport have been previously developed and can be used in the practiceof the invention. The vectors can be used with any pharmaceuticallyacceptable carrier for ease of administration and handling.

For purposes of intramuscular injection, solutions in an adjuvant suchas sesame or peanut oil or in aqueous propylene glycol can be employed,as well as sterile aqueous solutions. Such aqueous solutions can bebuffered, if desired, and the liquid diluent first rendered isotonicwith saline or glucose. Solutions of the chimeric or rHBoV vector as afree acid (DNA contains acidic phosphate groups) or a pharmacologicallyacceptable salt can be prepared in water suitably mixed with asurfactant such as hydroxypropylcellulose. A dispersion of viralparticles can also be prepared in glycerol, liquid polyethylene glycolsand mixtures thereof and in oils. Under ordinary conditions of storageand use, these preparations contain a preservative to prevent the growthof microorganisms. In this connection, the sterile aqueous mediaemployed are all readily obtainable by standard techniques well-known tothose skilled in the art.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of a dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal andthe like. In many cases it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the chimericor rHBoV vector in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the sterilized active ingredient into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the methods ofpreparation include but are not limited to vacuum drying and the freezedrying technique which yield a powder of the active ingredient plus anyadditional desired ingredient from the previously sterile-filteredsolution thereof.

For purposes of topical administration, dilute sterile, aqueoussolutions (usually in about 0.1% to 5% concentration), otherwise similarto the above parenteral solutions, are prepared in containers suitablefor incorporation into a transdermal patch, and can include knowncarriers, such as pharmaceutical grade di methylsulfoxide (DMSO).

Of interest is the correction of the genetic defect of cystic fibrosis,by supplying a properly functioning cystic fibrosis transmembraneconductance regulator (CFTR) to the airway epithelium. Thus, the use ofchimeric or rHBoV vectors encoding native CFTR protein, and mutants andfragments thereof, is envisioned.

Compositions of this invention may be used in vivo as well as ex vivo.In vivo gene therapy comprises administering the vectors of thisinvention directly to a subject. Pharmaceutical compositions can besupplied as liquid solutions or suspensions, as emulsions, or as solidforms suitable for dissolution or suspension in liquid prior to use. Foradministration into the respiratory tract, one mode of administration isby aerosol, using a composition that provides either a solid or liquidaerosol when used with an appropriate aerosolubilizer device. Anothermode of administration into the respiratory tract is using a flexiblefiberoptic bronchoscope to instill the vectors. Typically, the viralvectors are in a pharmaceutically suitable pyrogen-free buffer such asRinger's balanced salt solution (pH 7.4). Although not required,pharmaceutical compositions may optionally be supplied in unit dosageform suitable for administration of a precise amount.

An effective amount of virus is administered, depending on theobjectives of treatment. An effective amount may be given in single ordivided doses. Where a low percentage of transduction can cure a geneticdeficiency, then the objective of treatment is generally to meet orexceed this level of transduction. In some instances, this level oftransduction can be achieved by transduction of only about 1 to 5% ofthe target cells, but is more typically 20% of the cells of the desiredtissue type, usually at least about 50%, at least about 80%, at leastabout 95%, or at least about 99% of the cells of the desired tissuetype. As a guide, the number of vector particles present in a singledose given by bronchoscopy will generally be at least about 1×10¹²,e.g., about 1×10¹³, 1×10¹⁴, 1×10¹⁵ or 1×10¹⁶ particles, including bothDNAse-resistant and DNAse-susceptible particles. In terms ofDNAse-resistant particles, the dose will generally be between 1×10¹² and1×10¹⁶ particles, more generally between about 1×10¹² and 1×10¹⁵particles. The treatment can be repeated as often as every two or threeweeks, as required, although treatment once in 180 days may besufficient.

To confirm the presence of the desired DNA sequence in the host cell, avariety of assays may be performed. Such assays include, for example,“molecular biological” assays well known to those of skill in the art,such as Southern and Northern blotting, RT-PCR and PCR; “biochemical”assays, such as detecting the presence of a polypeptide expressed from agene present in the vector, e.g., by immunological means(immunoprecipitation, immunoaffinity columns, ELISAs and Western blots)or by any other assay useful to identify the presence and/or expressionof a particular nucleic acid molecule falling within the scope of theinvention.

To detect and quantitate RNA produced from introduced DNA segments,RT-PCR may be employed. In this application of PCR, it is firstnecessary to reverse transcribe RNA into DNA, using enzymes such asreverse transcriptase, and then through the use of conventional PCRtechniques amplify the DNA. In most instances PCR techniques, whileuseful, will not demonstrate integrity of the RNA product. Furtherinformation about the nature of the RNA product may be obtained byNorthern blotting. This technique demonstrates the presence of an RNAspecies and gives information about the integrity of that RNA. Thepresence or absence of an RNA species can also be determined using dotor slot blot Northern hybridizations. These techniques are modificationsof Northern blotting and only demonstrate the presence or absence of anRNA species.

While Southern blotting and PCR may be used to detect the DNA segment inquestion, they do not provide information as to whether the DNA segmentis being expressed. Expression may be evaluated by specificallyidentifying the polypeptide products of the introduced DNA sequences orevaluating the phenotypic changes brought about by the expression of theintroduced DNA segment in the host cell.

Thus, the effectiveness of the genetic alteration can be monitored byseveral criteria, including analysis of physiological fluid samples,e.g., urine, plasma, serum, blood, cerebrospinal fluid or nasal or lungwashes. Samples removed by biopsy or surgical excision may be analyzedby in situ hybridization, PCR amplification using vector-specificprobes, RNAse protection, immunohistology, or immunofluorescent cellcounting. When the vector is administered by bronchoscopy, lung functiontests may be performed, and bronchial lavage may be assessed for thepresence of inflammatory cytokines. The treated subject may also bemonitored for clinical features, and to determine whether the cellsexpress the function intended to be conveyed by the therapeutic orprophylactic polynucleotide.

The decision of whether to use in vivo or ex vivo therapy, and theselection of a particular composition, dose, and route of administrationwill depend on a number of different factors, including but not limitedto features of the condition and the subject being treated. Theassessment of such features and the design of an appropriate therapeuticor prophylactic regimen is ultimately the responsibility of theprescribing physician.

The foregoing description provides, inter alia, methods for generatinghigh titer preparations of recombinant chimeric viruses or rHBoV thatare substantially free of helper virus (e.g., adenovirus) and cellularproteins. It is understood that variations may be applied to thesemethods by those of skill in this art without departing from the spiritof this invention.

IV. Agents Useful in the Practice of the Invention

Classes of agents useful in the invention include but are not limited toantibiotics, chemotherapeutics, e.g., anthracyclines, proteasomemodulators, lipid lowering agents, mucolytic agents, and food additives.Exemplary agents include proteasome inhibitors (Wagner et al., 2002;Young et al., 2000; Seisenberger et al., 2001), as well as agents thatmodulate the proteosome and ubiquitin pathways, e.g., bind toproteosomes and/or modulate the activity of proteosomes, ubiquitin,ubiquitin carrier protein, or ubiquitin ligase. Examples of these agentsinclude without limitation antibiotics, e.g., epoxomicin, lipid loweringdrugs, e.g., simvastatin, food additives, e.g., tannic acid, andchemotherapeutics, e.g., cisplatin, anthracyclines such as doxorubicin,and camptothecin. In one embodiment, the agent is LLnL (MG101), Z-LLL(MG132), bortezomib (Velcade), epoxomicin, doxorubicin, doxil,daunorubicin, idarubicin, epirubicin, aclarubicin, simvastatin, tannicacid, camptothecin, or cisplatin.

In one embodiment, the agent is a compound of formula (I):R₁-A-(B)_(n)—C wherein R₁ is an N-terminal amino acid blocking group;each A and B is independently an amino acid; C is an amino acid whereinthe terminal carboxy group has been replaced by a formyl (CHO) group;and n is 0, 1, 2, or 3; or a pharmaceutically acceptable salt thereof.In one embodiment, R₁ is (C₁-C₁₀)alkanoyl. In one embodiment, R₁ isacetyl or benzyloxycarbonyl. In one embodiment, ach A and B isindependently alanine, arginine, glycine, isoleucine, leucine, valine,nor-leucine or nor-valine. In one embodiment, each A and B isisoleucine. In one embodiment, C is alanine, arginine, glycine,isoleucine, leucine, valine, nor-leucine or nor-valine, wherein theterminal carboxy group has been replaced by a formyl (CHO) group. In oneembodiment, C is nor-leucine or nor-valine, wherein the terminal carboxygroup has been replaced by a formyl (CHO) group. In one embodiment, R₁is (C₁-C₁₀)alkanoyl or benzyloxycarbonyl; A and B are each isoleucine; Cis nor-leucine or nor-valine, wherein the terminal carboxy group hasbeen replaced by a formyl (CHO) group; and N is 1.

Another example of an agent is a compound of formula (II):

wherein

R₂ is an N-terminal amino acid blocking group;

R₃, R₄, and R₅ are each independently hydrogen, (C₁-C₁₀)alkyl, aryl oraryl(C₁-C₁₀)alkyl; and

R₆, R₇, and R₈ are each independently hydrogen, (C₁-C₁₀)alkyl, aryl oraryl(C₁-C₁₀)alkyl; or a pharmaceutically acceptable salt thereof.

In yet another example, an agent useful in the methods is a compound offormula (III): R-A-A₁-R₁

wherein R is hydrogen, an amino acid, or a peptide, wherein theN-terminus amino acid can optionally be protected at the amino groupwith acetyl, acyl, trifluoroacetyl, or benzyloxycarbonyl; A is an aminoacid or a direct bond; A₁ is an amino acid; and

R₁ is hydroxy or an amino acid, wherein the C-terminus amino acid canoptionally be protected at the carboxy group with (C₁-C₆)alkyl, phenyl,benzyl ester or amide (e.g., C(═O)NR₂, wherein each R is independentlyhydrogen or (C₁-C₆)alkyl);

or a pharmaceutically acceptable salt thereof.

In one embodiment, the agent is H-Leu-Ala-OH, H—His-Ala-OH, or acombination thereof.

V. Dosages, Formulations and Routes of Administration of the Agents ofthe Invention

Administration of the agents identified in accordance with the presentinvention may be continuous or intermittent, depending, for example,upon the recipient's physiological condition, whether the purpose of theadministration is therapeutic or prophylactic, and other factors knownto skilled practitioners. The administration of the agents of theinvention may be essentially continuous over a preselected period oftime or may be in a series of spaced doses. Both local and systemicadministration is contemplated. When the agents of the invention areemployed for prophylactic purposes, agents of the invention are amenableto chronic use, e.g., by systemic administration.

One or more suitable unit dosage forms comprising the agents of theinvention, which, as discussed below, may optionally be formulated forsustained release, can be administered by a variety of routes includingoral, or parenteral, including by rectal, transdermal, subcutaneous,intravenous, intramuscular, intraperitoneal, intrathoracic,intrapulmonary and intranasal routes. For example, for administration tothe liver, intravenous administration may be preferred. Foradministration to the lung, airway administration may be preferred. Theformulations may, where appropriate, be conveniently presented indiscrete unit dosage forms and may be prepared by any of the methodswell known to pharmacy. Such methods may include the step of bringinginto association the agent with liquid carriers, solid matrices,semi-solid carriers, finely divided solid carriers or combinationsthereof, and then, if necessary, introducing or shaping the product intothe desired delivery system.

When the agents of the invention are prepared for oral administration,they may be combined with a pharmaceutically acceptable carrier, diluentor excipient to form a pharmaceutical formulation, or unit dosage form.The total active ingredients in such formulations comprise from 0.1 to99.9% by weight of the formulation. By “pharmaceutically acceptable” itis meant the carrier, diluent, excipient, and/or salt must be compatiblewith the other ingredients of the formulation, and not deleterious tothe recipient thereof. The active ingredient for oral administration maybe present as a powder or as granules; as a solution, a suspension or anemulsion; or in achievable base such as a synthetic resin for ingestionof the active ingredients from a chewing gum. The active ingredient mayalso be presented as a bolus, electuary or paste.

Pharmaceutical formulations containing the agents of the invention canbe prepared by procedures known in the art using well known and readilyavailable ingredients. For example, the agent can be formulated withcommon excipients, diluents, or carriers, and formed into tablets,capsules, suspensions, powders, and the like. Examples of excipients,diluents, and carriers that are suitable for such formulations includethe following fillers and extenders such as starch, sugars, mannitol,and silicic derivatives; binding agents such as carboxymethyl cellulose,HPMC and other cellulose derivatives, alginates, gelatin, andpolyvinyl-pyrrolidone; moisturizing agents such as glycerol;disintegrating agents such as calcium carbonate and sodium bicarbonate;agents for retarding dissolution such as paraffin; resorptionaccelerators such as quaternary ammonium compounds; surface activeagents such as cetyl alcohol, glycerol monostearate; adsorptive carrierssuch as kaolin and bentonite; and lubricants such as talc, calcium andmagnesium stearate, and solid polyethyl glycols.

For example, tablets or caplets containing the agents of the inventioncan include buffering agents such as calcium carbonate, magnesium oxideand magnesium carbonate. Caplets and tablets can also include inactiveingredients such as cellulose, pregelatinized starch, silicon dioxide,hydroxy propyl methyl cellulose, magnesium stearate, microcrystallinecellulose, starch, talc, titanium dioxide, benzoic acid, citric acid,corn starch, mineral oil, polypropylene glycol, sodium phosphate, andzinc stearate, and the like. Hard or soft gelatin capsules containing anagent of the invention can contain inactive ingredients such as gelatin,microcrystalline cellulose, sodium lauryl sulfate, starch, talc, andtitanium dioxide, and the like, as well as liquid vehicles such aspolyethylene glycols (PEGs) and vegetable oil. Moreover, enteric coatedcaplets or tablets of an agent of the invention are designed to resistdisintegration in the stomach and dissolve in the more neutral toalkaline environment of the duodenum.

The agents of the invention can also be formulated as elixirs orsolutions for convenient oral administration or as solutions appropriatefor parenteral administration, for instance by intramuscular,subcutaneous or intravenous routes.

The pharmaceutical formulations of the agents of the invention can alsotake the form of an aqueous or anhydrous solution or dispersion, oralternatively the form of an emulsion or suspension.

Thus, the therapeutic agent may be formulated for parenteraladministration (e.g., by injection, for example, bolus injection orcontinuous infusion) and may be presented in unit dose form in ampules,pre-filled syringes, small volume infusion containers or in multi-dosecontainers with an added preservative. The active ingredients may takesuch forms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredients may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

These formulations can contain pharmaceutically acceptable vehicles andadjuvants which are well known in the prior art. It is possible, forexample, to prepare solutions using one or more organic solvent(s) thatis/are acceptable from the physiological standpoint, chosen, in additionto water, from solvents such as acetone, ethanol, isopropyl alcohol,glycol ethers such as the products sold under the name “Dowanol”,polyglycols and polyethylene glycols, C₁-C₄ alkyl esters of short-chainacids, e.g., ethyl or isopropyl lactate, fatty acid triglycerides suchas the products marketed under the name “Miglyol”, isopropyl myristate,animal, mineral and vegetable oils and polysiloxanes.

The compositions according to the invention can also contain thickeningagents such as cellulose and/or cellulose derivatives. They can alsocontain gums such as xanthan, guar or carbo gum or gum arabic, oralternatively polyethylene glycols, bentones and montmorillonites, andthe like.

It is possible to add, if necessary, an adjuvant chosen fromantioxidants, surfactants, other preservatives, film-forming,keratolytic or comedolytic agents, perfumes and colorings. Also, otheractive ingredients may be added, whether for the conditions described orsome other condition.

For example, among antioxidants, t-butylhydroquinone, butylatedhydroxyanisole, butylated hydroxytoluene and α-tocopherol and itsderivatives may be mentioned. The galenical forms chiefly conditionedfor topical application take the form of creams, milks, gels, dispersionor microemulsions, lotions thickened to a greater or lesser extent,impregnated pads, ointments or sticks, or alternatively the form ofaerosol formulations in spray or foam form or alternatively in the formof a cake of soap.

Additionally, the agents are well suited to formulation as sustainedrelease dosage forms and the like. The formulations can be soconstituted that they release the active ingredient only or for instancein a particular part of the intestinal or respiratory tract, possiblyover a period of time. The coatings, envelopes, and protective matricesmay be made, for example, from polymeric substances, such aspolylactide-glycolates, liposomes, microemulsions, microparticles,nanoparticles, or waxes. These coatings, envelopes, and protectivematrices are useful to coat indwelling devices, e.g., stents, catheters,peritoneal dialysis tubing, and the like.

The agents of the invention can be delivered via patches for transdermaladministration. See U.S. Pat. No. 5,560,922 for examples of patchessuitable for transdermal delivery of an agent. Patches for transdermaldelivery can comprise a backing layer and a polymer matrix which hasdispersed or dissolved therein an agent, along with one or more skinpermeation enhancers. The backing layer can be made of any suitablematerial which is impermeable to the agent. The backing layer serves asa protective cover for the matrix layer and provides also a supportfunction. The backing can be formed so that it is essentially the samesize layer as the polymer matrix or it can be of larger dimension sothat it can extend beyond the side of the polymer matrix or overlay theside or sides of the polymer matrix and then can extend outwardly in amanner that the surface of the extension of the backing layer can be thebase for an adhesive means. Alternatively, the polymer matrix cancontain, or be formulated of, an adhesive polymer, such as polyacrylateor acrylate/vinyl acetate copolymer. For long-term applications it mightbe desirable to use microporous and/or breathable backing laminates, sohydration or maceration of the skin can be minimized.

Examples of materials suitable for making the backing layer are films ofhigh and low density polyethylene, polypropylene, polyurethane,polyvinylchloride, polyesters such as poly(ethylene phthalate), metalfoils, metal foil laminates of such suitable polymer films, and thelike. The materials used for the backing layer may be laminates of suchpolymer films with a metal foil such as aluminum foil. In suchlaminates, a polymer film of the laminate will usually be in contactwith the adhesive polymer matrix.

The backing layer can be any appropriate thickness, which will providethe desired protective and support functions. A suitable thickness willbe from about 10 to about 200 microns.

Generally, those polymers used to form the biologically acceptableadhesive polymer layer are those capable of forming shaped bodies, thinwalls or coatings through which agents can pass at a controlled rate.Suitable polymers are biologically and pharmaceutically compatible,nonallergenic and insoluble in and compatible with body fluids ortissues with which the device is contacted. The use of soluble polymersis to be avoided since dissolution or erosion of the matrix by skinmoisture would affect the release rate of the agents as well as thecapability of the dosage unit to remain in place for convenience ofremoval.

Exemplary materials for fabricating the adhesive polymer layer includepolyethylene, polypropylene, polyurethane, ethylene/propylenecopolymers, ethylene/ethylacrylate copolymers, ethylene/vinyl acetatecopolymers, silicone elastomers, especially the medical-gradepolydimethylsiloxanes, neoprene rubber, polyisobutylene, polyacrylates,chlorinated polyethylene, polyvinyl chloride, vinyl chloride-vinylacetate copolymer, crosslinked polymethacrylate polymers (hydrogel),polyvinylidene chloride, poly(ethylene terephthalate), butyl rubber,epichlorohydrin rubbers, ethylene vinyl alcohol copolymers,ethylene-vinyloxyethanol copolymers; silicone copolymers, for example,polysiloxane-polycarbonate copolymers, polysiloxane-polyethylene oxidecopolymers, polysiloxane-polymethacrylate copolymers,polysiloxane-alkylene copolymers (e.g., polysiloxane-ethylenecopolymers), polysiloxane-alkylenesilane copolymers (e.g.,polysiloxane-ethylenesilane copolymers), and the like; cellulosepolymers, for example methyl or ethyl cellulose, hydroxy propyl methylcellulose, and cellulose esters; polycarbonates;polytetrafluoroethylene; and the like.

A biologically acceptable adhesive polymer matrix may be selected frompolymers with glass transition temperatures below room temperature. Thepolymer may, but need not necessarily, have a degree of crystallinity atroom temperature. Cross-linking monomeric units or sites can beincorporated into such polymers. For example, cross-linking monomers canbe incorporated into polyacrylate polymers, which provide sites forcross-linking the matrix after dispersing the agent into the polymer.Known cross-linking monomers for polyacrylate polymers includepolymethacrylic esters of polyols such as butylene diacrylate anddimethacrylate, trimethylol propane trimethacrylate and the like. Othermonomers which provide such sites include allyl acrylate, allylmethacrylate, diallyl maleate and the like.

A plasticizer and/or humectant may be dispersed within the adhesivepolymer matrix. Water-soluble polyols are generally suitable for thispurpose. Incorporation of a humectant in the formulation allows thedosage unit to absorb moisture on the surface of skin which in turnhelps to reduce skin irritation and to prevent the adhesive polymerlayer of the delivery system from failing.

Agents released from a transdermal delivery system must be capable ofpenetrating each layer of skin. In order to increase the rate ofpermeation of an agent, a transdermal drug delivery system must be ablein particular to increase the permeability of the outermost layer ofskin, the stratum corneum, which provides the most resistance to thepenetration of molecules. The fabrication of patches for transdermaldelivery of agents is well known to the art.

For administration to the upper (nasal) or lower respiratory tract byinhalation, the agents of the invention are conveniently delivered froman insufflator, nebulizer or a pressurized pack or other convenientmeans of delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecomposition may take the form of a dry powder, for example, a powder mixof the agent and a suitable powder base such as lactose or starch. Thepowder composition may be presented in unit dosage form in, for example,capsules or cartridges, or, e.g., gelatine or blister packs from whichthe powder may be administered with the aid of an inhalator, insufflatoror a metered-dose inhaler.

For intra-nasal administration, the agent may be administered via nosedrops, a liquid spray, such as via a plastic bottle atomizer ormetered-dose inhaler. Typical of atomizers are the Mistometer (Wintrop)and the Medihaler (Riker).

The local delivery of the agents of the invention can also be by avariety of techniques which administer the agent at or near the site ofdisease. Examples of site-specific or targeted local delivery techniquesare not intended to be limiting but to be illustrative of the techniquesavailable. Examples include local delivery catheters, such as aninfusion or indwelling catheter, e.g., a needle infusion catheter,shunts and stents or other implantable devices, site specific carriers,direct injection, or direct applications.

For topical administration, the agents may be formulated as is known inthe art for direct application to a target area. Conventional forms forthis purpose include wound dressings, coated bandages or other polymercoverings, ointments, creams, lotions, pastes, jellies, sprays, andaerosols. Ointments and creams may, for example, be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agents. Lotions may be formulated with an aqueous or oily baseand will in general also contain one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents, thickeningagents, or coloring agents. The active ingredients can also be deliveredvia iontophoresis, e.g., as disclosed in U.S. Pat. Nos. 4,140,122;4,383,529; or 4,051,842. The percent by weight of an agent of theinvention present in a topical formulation will depend on variousfactors, but generally will be from 0.01% to 95% of the total weight ofthe formulation, and typically 0.1-25% by weight.

Drops, such as eye drops or nose drops, may be formulated with anaqueous or non-aqueous base also comprising one or more dispersingagents, solubiling agents or suspending agents. Liquid sprays areconveniently delivered from pressurized packs. Drops can be deliveredvia a simple eye dropper-capped bottle, or via a plastic bottle adaptedto deliver liquid contents dropwise, via a specially shaped closure.

The agent may further be formulated for topical administration in themouth or throat. For example, the active ingredients may be formulatedas a lozenge further comprising a flavored base, usually sucrose andacacia or tragacanth; pastilles comprising the composition in an inertbase such as gelatin and glycerin or sucrose and acacia; and mouthwashescomprising the composition of the present invention in a suitable liquidcarrier.

The formulations and compositions described herein may also containother ingredients such as antimicrobial agents, or preservatives.Furthermore, the active ingredients may also be used in combination withother agents, for example, bronchodilators.

The agents of this invention may be administered to a mammal alone or incombination with pharmaceutically acceptable carriers. As noted above,the relative proportions of active ingredient and carrier are determinedby the solubility and chemical nature of the compound, chosen route ofadministration and standard pharmaceutical practice.

The dosage of the present agents will vary with the form ofadministration, the particular compound chosen and the physiologicalcharacteristics of the particular patient under treatment. Generally,small dosages will be used initially and, if necessary, will beincreased by small increments until the optimum effect under thecircumstances is reached.

VI. Exemplary Embodiments

In one embodiment, the invention provides an isolated chimeric viruscomprising bocavirus capsid protein and a rAAV genome. In oneembodiment, the bocavirus capsid is a HBoV capsid. In one embodiment,the HBoV capsid is HBoV1, HBoV2, HBoV3 or HBoV4capsid. In oneembodiment, the genome comprises an expression cassette encoding aheterologous gene product, e.g., a therapeutic protein. In oneembodiment, the rAAV genome is a rAAV-2 genome. In one embodiment, therAAV genome is a rAAV-1, rAAV-3, rAAV-4, rAAV-5, rAAV-6, rAAV-7, rAAV-8or rAAV-9 genome. In one embodiment, the rAAV genome is derived from anon-human species of AAV. In one embodiment, the expression cassetteincludes a promoter that is expressed in ciliated airway epithelailcells, e.g., a FOXJ1 promoter. In one embodiment, the genome includes atrans-splicing domain. The gene product encoded by the viral genome maybe a viral, bacterial, tumor or fungal antigen. In one embodiment, thetransgene encodes a neutralizing antibody or an antigen binding fragmentthereof. In one embodiment, the gene product is cystic fibrosistransmembrane conductance regulator, β-globin, γ-globin, tyrosinehydroxylase, glucocerebrosidase, aryl sulfatase A, factor VIII,dystrophin, alpha 1-antitrypsin, surfactant protein SP-D, SP-A or SP-C,erythropoietin, RSV protein, HBoV protein, influenza virus protein, SARSprotein, a cytokine, e.g., IFN-alpha, IFN-gamma, TNF, IL-1, IL-17, orIL-6. Also provided is a method to express a heterologous gene productin mammalian cells which employs the isolated chimeric virus to infectcells in an amount effective to express a heterologous gene product,e.g., a therapeutic gene product, a catalytic RNA, a prophylactic geneproduct, a polypeptide or peptide. In one embodiment, the virus isisolated from insect cells or mammalian cells. Further provided is amethod to enhance chimeric virus transduction of a mammalian cell. Themethod includes contacting a mammalian cell with an isolated chimericvirus comprising human bocavirus capsid protein and a rAAV genomecomprising a transgene encoding a heterologous gene product and at leastone agent in an amount effective to additively or synergisticallyenhance rAAV transduction. In one embodiment, the mammalian cell is amammalian lung cell. In one embodiment, the agent is a porteasomeinhibitor, chemotherapeutic, a lipid lowering agent, a mucolytic agent,an antibiotic or a food additive.

The isolated chimeric virus may be employed in a method to inhibit ortreat a condition associated with aberrant expression of an endogenousgene product. The method includes contacting a mammal at risk of orhaving the condition, with an effective amount of the isolated chimericvirus comprising human bocavirus capsid proteins and a rAAV genome,wherein the rAAV genome comprises a transgene encoding at least aportion of a functional gene product, the expression of which in themammal inhibits or treats at least one symptom of the condition. In oneembodiment, the gene product is cystic fibrosis transmembraneconductance regulator, β-globin, γ-globin, tyrosine hydroxylase,glucocerebrosidase, aryl sulfatase A, factor VIII, dystrophin, alpha1-antitrypsin, surfactant protein SP-D, SP-A or SP-C, erythropoietin,RSV protein, HBoV protein, influenza virus protein, SARS protein, acytokine, e.g., IFN-alpha, IFN-gamma, TNF, IL-1, IL-17, or IL-6. In oneembodiment, the mammal is further contacted with at least one proteasomeinhibitor, a chemotherapeutic, a lipid lowering agent, an antibiotic ora food additive in an amount that enhances transduction. In oneembodiment, the at least one agent is LLnL (MG101), Z-LLL (MG132),bortezomib (Velcade), epoxornicin, doxorubicin, doxil, daunorubicin,idarubicin, epirubicin, aclarubicin, simvastatin, tannic acid,camptothecin, or cisplatin. An agent may be employed in a method toenhance virus transduction of a mammalian cell. A mammalian cell iscontacted with a chimeric virus comprising a human bocavirus capsidprotein and a rAAV genome and an agent in an amount effective to enhancetransduction of the virus relative to a mammalian cell that is notcontacted with the agent. In one embodiment, the agent is a proteasomeinhibitor. Further provided is a method to enhance the expression of atransgene in a mammalian cell, where a mammalian cell is contacted withan amount of an agent that is a proteasome inhibitor and a chimericvirus comprising a human bocavirus capsid protein and a rAAV genomecomprising the transgene. The amount of the agent enhances transductionof the rAAV, thereby enhancing expression of the transgene, relative toa mammalian cell that is not contacted with the agent.

Also provided is a method in which a mammal subjected to viral genetherapy with an isolated chimeric virus comprising human bocaviruscapsid proteins and a rAAV genome, wherein the genome comprises atransgene the expression of which in the mammal is therapeutic, isadministered an agent that is a proteasome inhibitor in an amounteffective to enhance expression of the transgene in the cells of themammal relative to cells in a mammal that are not contacted with theagent. In one embodiment, the rAAV encodes a therapeutic peptide or atherapeutic polypeptide. In one embodiment, the cell or mammal iscontacted with the agent before the cell or mammal is contacted with thevirus. In one embodiment, the cell or mammal is contacted with the virusbefore the cell or mammal is contacted with the agent. In oneembodiment, the cell or mammal is contacted with the virus and agentconcurrently. In one embodiment, the agent and the virus areadministered to the lung. In one embodiment, the the virus is orallyadministered. In one embodiment, the virus is nasally administered. Inone embodiment, the virus is administered to a blood vessel.

Further provided is a method to immunize a mammal. The method includesadministering to a mammal an isolated chimeric virus comprising humanbocavirus capsid proteins and a rAAV genome encoding a prophylactic geneproduct in an amount effective to prevent or inhibit microbial infectionor replication. In one embodiment, the gene product is an antigen of avirus, bacteria, fungus or parasite. In one embodiment, the gene productis a neutralizing antibody against a virus, bacteria, fungus orparasite.

The invention provides an isolated rHBoV comprising human bocaviruscapsid protein and a recombinant HBoV genome. In one embodiment, thegenome comprises an expression cassette encoding a heterologous geneproduct. In one embodiment, the gene product encodes a therapeuticprotein. In one embodiment, the gene product is a viral, bacterial,tumor or fungal antigen. In one embodiment, the gene product is cysticfibrosis transmembrane conductance regulator, β-globin, γ-globin,tyrosine hydroxylase, glucocerebrosidase, aryl sulfatase A, factor VIII,dystrophin, alpha 1-antitrypsin, surfactant protein SP-D, SP-A or SP-C,erythropoietin, HBoV protein, influenza virus protein, RSV protein, SARSprotein, or a cytokine, e.g., IFN-alpha, IFNγ, TNF, IL-1, IL-17, orIL-6. The isolated rHBoV may be employed to express a heterologous geneproduct in mammalian cells. The cells are infected with the virus in anamount effective to express the heterologous gene product, e.g., atherapeutic gene product, a catalytic RNA, a prophylactic gene product,a polypeptide or peptide. Also provided is a method to enhance chimericvirus transduction of a mammalian cell. The method includes contacting amammalian cell with an isolated rHBoV comprising human bocavirus capsidprotein and a rHBoV genome comprising a transgene encoding aheterologous gene product and at least one agent in an amount effectiveto additively or synergistically enhance transduction. In oneembodiment, the mammalian cell is a mammalian lung cell. In oneembodiment, the agent is a porteasome inhibitor, chemotherapeutic, alipid lowering agent, an antibiotic or a food additive.

Further provided is a method to inhibit or treat a condition associatedwith aberrant expression of an endogenous gene product. The methodincludes contacting a mammal at risk of or having the condition, with aneffective amount of an isolated rHBoV comprising human bocavirus capsidproteins and a rHBoV genome, wherein the rHBoV genome comprises atransgene encoding at least a portion of a functional gene product, theexpression of which in the mammal inhibits or treats at least onesymptom of the condition. In one embodiment, the gene product is cysticfibrosis transmembrane conductance regulator, β-globin, γ-globin,tyrosine hydroxylase, glucocerebrosidase, aryl sulfatase A, factor VIII,dystrophin, alpha 1-antitrypsin, surfactant protein SP-D, SP-A or SP-C,erythropoietin, HBoV protein, influenza virus protein, RSV protein, SARSprotein, IFN-alpha, TNF, IL-1, IL-17, or IL-6. In one embodiment,further comprising contacting the mammal with at least one proteasomeinhibitor, a chemotherapeutic, a lipid lowering agent, a mucolyticagent, an antibiotic or a food additive in a an amount that enhancestransduction. In one embodiment, the at agent is LLnL (MG101), Z-LLL(MG132), bortezomib (Velcade), epoxomicin, doxorubicin, doxil,daunorubicin, idarubicin, epirubicin, aclarubicin, simvastatin, tannicacid, camptothecin, or cisplatin.

In addition the invention provides a method to enhance virustransduction of a mammalian cell, where a mammalian cell is contactedwith a rHBoV comprising a human bocavirus capsid protein and a rHBoVgenome and an agent in an amount effective to enhance transduction ofthe virus relative to a mammalian cell that is not contacted with theagent, e.g., the agent is a proteasome inhibitor. The agent and rHBoVmay be employed to a method to enhance the expression of a transgene ina mammalian cell. The method includes contacting the mammalian cell withan amount of an agent that is a proteasome inhibitor and a rHBoVcomprising a human bocavirus capsid protein and a rHBoV genomecomprising the transgene, wherein the amount enhances transduction,thereby enhancing expression of the transgene, relative to a mammaliancell that is not contacted with the agent. An agent may be administeredto a mammal subjected to viral gene therapy with an isolated rHBoVcomprising human bocavirus capsid proteins and a rHBoV genome, whereinthe genome comprises a transgene the expression of which in the mammalis therapeutic. The agent may be a proteasome inhibitor and isadministered in an amount effective to enhance expression of thetransgene in the cells of the mammal relative to cells in a mammal thatare not contacted with the agent. In one embodiment, the rHBoV encodes atherapeutic peptide or a therapeutic polypeptide. In one embodiment, thecell or mammal is contacted with the agent before the cell is contactedwith the virus. In one embodiment, the cell or mammal is contacted withthe virus before the cell is contacted with the agent. In oneembodiment, the cell or mammal is contacted with the virus and agentconcurrently. In one embodiment, the agent and the virus areadministered to the lung. In one embodiment, the virus is orallyadministered. In one embodiment, the virus is nasally administered. Inone embodiment, the virus is administered to a blood vessel.

An isolated rHBoV comprising human bocavirus capsid proteins and a rHBoVgenome encoding a prophylactic gene product may be employed in a methodto immunize a mammal. The virus is administered to a mammal in an amounteffective to prevent or inhibit microbial infection or replication.

Further provided is a method to immunize a mammal, includingadministering to a mammal an isolated chimeric virus comprising humanbocavirus capsid proteins and a rAAV genome in an amount effective toprevent or inhibit HBoV infection or replication. In one embodiment, thechimeric virus is administered to the lung. Also provided is a vaccinecomprising the chimeric virus.

Further provided is a method to immunize a mammal, comprising:administering to a mammal an isolated rHBoV comprising human bocaviruscapsid proteins and a rHBoV genome in an amount effective to prevent orinhibit HBoV infection or replication. In one embodiment, the chimericvirus is administered to the lung. Also provided is a vaccine comprisingthe virus.

The invention will be further described by the following non-limitingexamples.

Example 1

Materials and Methods

Cell Culture

Cell lines and primary cells. Human embryonic kidney 293 (HEK293) cells(CRL-1573), HeLa (CCL-2), MDCK (CCL-34), MRC-5 (CCL-171), LLC-MK2(CCL-7), and Vero-E6 (CRL-1586) were obtained from American Type CultureCollection (ATCC, Manassas, Va.), and were cultured in Dulbecco'sModified Eagle Medium (DMEM) with 10% fetal calf serum (FCS). The celllines originating from human airway epithelial cells are A549 (ATCCCCL-185). BEAS-2B (ATCC CRL-9609), 16HBE14o- (obtained from Dr. DieterGruenert), as well as NuLi-1 and CuFi-8 (Tissue and Cell Culture Core,Center for Gene Therapy, University of Iowa). NuLi-1 and CuFi-8 wereimmortalized from normal and cystic fibrosis human primary airway cells,respectively, by expressing hTERT and HPV E6/E7 genes (Zabner et al.,2003). Primary Clonetics normal human bronchial/tracheal epithelialcells (NHBE) were purchased from Lonza (Walkersville, Md.). Cells werecultured in media following instructions provided by the supplier.

Human airway epithelium cultures. Polarized primary HAE, termed asprimary B-HAE, was generated by growing isolated human airway(tracheobronchial) epithelial cells (three HAE cultures were generatedfrom different donors) on collagencoated, semipermeable membrane inserts(0.6 cm2, Millicell-PCF; Millipore, Billerica, Mass.), and then allowingthem to differentiate at an air-liquid interface (ALI); this was carriedout at the Tissue and Cell Culture Core of the Center for Gene Therapy,University of Iowa (Zabner et al., 2003; Karp et al., 2002; Yan et al.,2004; Yan et al., 2006). After 3-4 weeks of culture at an ALI, thepolarity of the HAE was determined based on the transepithelialelectrical resistance (TEER) using an epithelial Volt-Ohm Meter(Millipore) and the relationship to infectibility was assessed; a valueof over 1,000 V·cm2 was required for HBoV1 infection. CuFi- and NuLi-HAEwere generated following the same method as above, but using theimmortalized airway epithelial cell lines, CuFi-8 and NuLi-1,respectively. The primary B-, CuFi-, and NuLi-HAE were cultured,differentiated and maintained in (50%:50%) DMEM:F12 medium containing 2%Ultroser G (Pall BioSepra, Cergy-Staint-Christophe, France).

Isolation of Virus and Extraction of Viral DNA

A nasopharyngeal aspirate was obtained from a child withcommunity-acquired pneumonia in Salvador, Brazil, who had an acute HBoV1infection (seroconversion, viraemia, and over 104 gc of HBoV1 per ml ofaspirate) (Nascimento-Carvalho et al., 2012). Viral DNA was extractedaccording to a method described in Kantola et al. (2010).

Primers Used and Sequence Amplification by the Polymerase Chain Reaction(PCR)

The sequence of the head-to-tail junction of the HBoV1 episome suggeststhat HBoV LEH and REH share similarities both in structure and sequencewith that of the BPV LEH and MVC REH, respectively (Sun et al., 2009;Lusebrink et al., 2011). The Phusion high fidelity PCR kit (NEB,Ipswich, Mass.) was used following the manufactures' instructions, toamplify the left-end hairpin (LEH) and the right-end hairpin (REH) ofHBoV1. Briefly, the DNA denaturation at 98° C. for 30 seconds wasfollowed by 35 cycles of: denaturing at 98° C. for 10 seconds; annealingat 55° C. for 15 seconds; and extension at 72° C. for 30 seconds.Following the final cycle, extension was continued at 72° C. for 10minutes. The PCR products were analyzed by electrophoresis in a 2%agarose gel. DNA bands were extracted using the QIAquick gel extractionkit (Qiagen, Valencia, Calif.), and the extracted DNA was directlysequenced at MCLAB (South San Francisco, Calif.), using primerscomplementary to the extended sequences on the forward and reverseamplification primers. PCR-generated DNA was cloned in pGEM-T vector(Promega, Madison, Wis.), and DNAs isolated from cultures of individualclones were subsequently sequenced.

Construction of a Full-Length HBoV1 Clone and its Mutants

Construction of the pBB Vector.

A pBBSmaI vector was constructed by inserting a linker of59-SalI-SacII-KpnI-SmaIApaI-SphI-KpnI-HindIII-XhoI-39 into a vectorbackbone (pProEX HTb vector; Invitrogen) generated from the B19Vinfectious clone pM20 (Zhi et al., 2004) by removing all of the B19Vsequence (SalI-digestion). All cloning work was carried out in theEscherichia coli strain of Sure 2 (Agilent, La Jolla, Calif.). All thenucleotide numbers of HBoV1 refer to the HBoV1 full-length genome(GenBank accession no.:JQ923422).

Cloning of the Left-End Hairpin.

The DNA fragment SalI-BglII-nt93-518(BspEI)-576-XhoI-HindIII (containingthe HBoV1 sequence nt 93-576), was amplified from the viral DNA andinserted into SalI/HindIII-digested pBBSmaI, to produce pBB2.1. AnotherDNA, SalI-nt1-86-BclI (containing HBoV1 nt 1-86 sequence), wassynthesized according to the LEH sequence obtained, and placed betweenthe SalI and BglII sites in pBB2.1, with ligation of the BclI and BglIIsites reproducing the HBoV1 sequence nt 87-92. The resultant plasmidharboring the 59 HBoV1 nt 1-576 sequence with an intact LEH isdesignated pBB-LEH.

Cloning of the Right-End Hairpin.

The DNA fragment SalI-nt4097-4139(BglII)-5427(KasI)-ApaI (containing theHBoV1 nt 4097-5427 sequence) was amplified from viral DNA and insertedinto Sail/ApaI-digested pBBSmaI, resulting in pBB2.2. Another DNAfragment, ApaI-nt5460(KasI)-5543-XhoI (containing HBoV1 nt 5460-5543sequence) was synthesized based on the REH sequence and placed betweenthe ApaI and HindIII sequences in pBB2.2, resulting in pBBREH(D5428-5459). The missing short fragment between the two KasI sitesencompassing nt 5428-5459 was recovered by a synthesized KasI linkerbased on the REH sequence and inserted into KasI-digestedpBB-REH(D5428-5459). The resultant plasmid harboring the 39 HBoV1 nt4097-5543 sequence with an intact REH is designated pBB-REH.

Cloning of the pIHBoV1.

The HBoV1 DNA fragment SalI-nt1-518(BspEI)-576-XhoI, which was obtainedfrom SalI/XhoI-digestion of pBB-LEH, was ligated into SalI-digestedpBB-REH, resulting in pBB-LEH(BspEI/BglII)REH. The larger fragmentproduced by digestion of this plasmid with BspEI/BglII was ligated tothe HBoV1 DNA fragment nt 518(BspEI)-4139(BglII), which was amplifiedfrom the viral DNA. The final construct containing the full-length HBoV1(nt 1-5543) was designated pIHBoV1.

Construction of pIHBoV1 Mutants.

pIHBoV1NS1(2) and pi HBoV1NP1(2) were constructed by mutating HBoV1 nt542 from T to A, and nt 2588 from G to A, resulting in stop codons thatlead to early termination of the NS1 and NP1 ORFs, respectively.Similarly, pIHBoV1VP1(2) and pIHBoV1 VP2(2) were generated by mutatingHBoV1 nt 3205 from T to A, and nt 3540 from T to G, disrupting VP1 andVP2 ORFs, respectively.

Transfection

Cells grown in 60-mm dishes were transfected with 2 mg of plasmid; theLipofectamine and Plus reagents (Invitrogen/Life Technologies, Carlsbad,Calif.) were used as described in Qiu et al. (2002). For some of thetransfection experiments, HEK293 cells were cotransfected with 2 mg ofpHelper plasmid (Agilent), which contains the adenovirus 5 (Ad5) Eta,E4orf6, and VA genes, or infected with adenovirus type 5 (Ad) at an MOIof 5 as described in Qiu et al. (2002).

Southern Blot Analysis

Low molecular weight (Hirt) DNA was extracted from transfected cells,digested with DpnI (or left undigested) and analyzed by Southernblotting as described in Qiu et al. (2006).

Western Blot Analysis

Cells were lysed, separated by SDS-8% polyacrylamide gel electrophoresis(PAGE), and blotted with antibodies as indicated as described in Liu etal. (2004).

Production and Purification of HBoV1

HEK293 cells were cultured on fifteen 150-mm plates in DMEM-10% FCS, andtransfected with 15 mg of pIHBoV1 per dish using LipoD293 (SignaGen,Gaithersburg, Md.). After being maintained for 48 hours at 5% CO₂ and37° C., the cells were collected, resuspended in 10 mL of phosphatebuffered saline, pH7.4 (PBS), and lysed by subjecting them to fourfreezing (−196° C.) and thawing (37° C.) cycles. The cell lysate wasthen spun at 10,000 rpm for 30 minutes. The supernatant was collectedand assessed on a continuous CsCl gradient. In brief, the density wasadjusted to 1.40 g/mL by adding CsCl, and the sample was loaded into an11-ml centrifuge tube and spun in a Sorvall TH641 rotor at 36,000 rpm,for 36 hours at 20° C.

Fractions of 550 mL (20 fractions) were collected with a Piston GradientFractionator (BioComp, Fredericton, NB, Canada), and the density of eachwas determined by an Abbe's Refractometer. Viral DNA was extracted fromeach fraction and quantified with respect to the number of HBoV1 gc,using HBoV1-specific qPCR as described below. Those fractions containingthe highest numbers of HBoV1 gc were dialyzed against PBS, and thenviewed by electron microscope and used to infect HAE cultures.

Observation by Electron Microscopy (EM)

The final purified virus preparation was concentrated by about 5-fold,and adsorbed for 1 minute on a 300-mesh copper EM grid coated with acarbon film, followed by washing with deionized water for 5 seconds andstaining with 1% uranyl acetate for 1 minute. The grid was air dried,and was inspected on a 200 kV Tecnai F20 G2 transmission electronmicroscope equipped with a field emission gun.

Virus Infection

Fully differentiated primary B- (each of the three distinct subtypes),CuFi- and NuLi-HAE were cultured in Millicell inserts (0.6 cm²;Millipore) and inoculated with 150 μL of purified HBoV1 (1×10⁷ gc/mL inphosphate buffered saline, pH7.4; PBS) from the apical surface (at amultiplicity of infection, MOI, of about 750 gc/cell; an average of2×10⁶ cells per insert). For each of the HAE, a 2 hour incubation wasfollowed by aspiration of the virus from the apical chamber and by threewashes of the cells with 200 mL of PBS to remove unbound virus. The HAEswere then further cultured at an ALI.

For conventional monolayer cells, cells cultured in chamber slides(Lab-Tek II; Nalge Nunc) were infected with purified HBoV1 at an MOI of1,000 gc/cell.

Immunofluorescence Analysis

After HBoV1 infection, ALI membranes were fixed with 3.7%paraformaldehyde in PBS at room temperature for 15 min. The fixedmembranes were cut into several small pieces, washed in PBS three timesfor 5 minutes, and permeabilized with 0.2% Triton X-100 for 15 minutesat room temperature. The membranes were then incubated with primaryantibody at a dilution of 1:100 in PBS with 2% FCS for 1 hour at 37° C.This was followed by incubation with a fluorescein isothiocyanate- orrhodamine-conjugated secondary antibody. Confocal images were taken withan Eclipse C1 Plus confocal microscope (Nikon, Melville, N.Y.)controlled by Nikon EZ-C₁ software. Primary antibodies used wereanti-(HBoV1) NS1, NP1 and VP1/2 antibodies, as reported in Chen et al.(2010).

For infected cells cultured in chamber slides, IF analysis was carriedout as previously described in Chen et al. (2010).

Quantitative PCR (qPCR) Analysis

Virus samples were collected from both the apical and basolateralsurfaces at multiple time points. Apical washing and harvesting wasperformed by adding 200 mL of PBS to the apical chamber, incubating thesamples for 10 minutes at 37° C. and 5% CO₂, and removing and storingthe 200 mL of PBS from the apical chamber. Thereafter, 50 mL of mediumwere collected from each basolateral chamber.

Aliquots (100 mL apical or 50 mL basolateral) of the samples wereincubated with 25 units of Benzonase (Sigma, St Louis, Mo.) for 2 hoursat 37° C., and then digested with 20 mL of proteinase K (15 mg/mL) at56° C. for 10 minutes. Viral DNA was extracted using QIAamp blood minikit (Qiagen), and eluted in 100 mL or 50 mL of deionized H₂O. Theextracted DNA was quantified with respect to the number of HBoV1 gc, bya qPCR method that has been used previously (see Lin et al., 2007).Briefly, the pskHBoV1 plasmid (Chen et al., 2010), which contains theHBoV1 sequence (nt 1-5299), was used as a control (1 gc=5.4610212 mg) toestablish a standard curve for absolute quantification with an AppliedBiosystems 7500 Fast system (Foster City, Calif.). The amplicon primersand the PrimeTime dual-labeled probe were designed by Primer Express(version 2.0.0; Applied Biosystems/Life Technologies) and synthesized atIDT Inc. (Coralville, Iowa). Their sequences are as follows (GenBank:JQ411251): forward primers, 5′-GCA CAG CCA CGT GAG GAA-3′ (SEQ ID NO:1;nt 2391 to 2408); reverse primer, 5′-TGG ACT CCC TTT TCT TTT GTA GGA-3′(SEQ ID NO:2; nt 2466 to 2443); and PrimeTime probe, 5′ 6FAM-TGA GCT CAGGGA ATA TGA AAG ACA AGC ATC G-3′ Iowa Black FQ (SEQ ID NO:3; nt 2411 to2441). Premix Ex Taq (Takara Bio USA, Madison, Wis.) was used for qPCRfollowing a standard protocol. 2.5 mL of extracted DNA was used in areaction volume of 25 mL.

Histology Analysis

On the last day of infection, the HAE on the Millicell inserts werewashed with PBS and fixed in 4% paraformaldehyde for about 30 minutes.The fixed membranes were cut into several small pieces, and washed withPBS three times. Each membrane fragment was transferred to 20% sucrosein a 15-mL conical tube and allowed to drop to the bottom; it was thenembedded vertically in cryoprotectant OCT in an orientation that enabledsectioning of the membrane perpendicular to the blade. Cryostat sectionswere cut at a thickness of 10 mm, placed onto slides, and stained withhematoxylin and eosin (H&E). Images were taken with a Nikon 80ifluorescence microscope at a magnification of ×60.

Results

The terminal hairpins of the HBoV1 genome are typical of those of thegenus Bocavirus A head-to-tail junction of an HBoV1 episome identifiedin an HBoV1-infected HAE (Schildgren et al., 2012; Lasebrink et al.,2011) was found to possess two sequences (3′-CGCGCGTA-5′ and3′-GATTAG-5′) identical to parts of the BPV1 left-end hairpin (LEH) (Sunet al., 2009; Chen et al., 1986). This finding suggested that the headsequence is part of the HBoV1 LEH. The head sequence was used as the 39end of a reverse primer (RHBoV1_LEH). Together with a forward primer(FHBoV1_nt1), which anchors the 39 end of the HBoV1 genome predictedfrom the BPV1 LEH, the hairpin of the LEH was amplified from a viral DNAextract (1.26108 gc/mL) prepared from a nasopharyngeal aspirate takenfrom an HBoV1-infected patient (HBoV1 Salvador1 isolate)(Nascimento-Carvalho et al., 2012). Only one specific DNA band wasdetected at approximately 150-bp.

Sequencing of this DNA revealed a novel sequence of the HBoV1 LEH.Because the LEHs of the prototype bocaviruses BPV1 and MVC areasymmetric (Sun et al., 2009; Chen et al., 1986), another PCR reactionwas set up with a forward primer located in the hairpin (FHBoV1_LEH) anda reverse primer targeting a sequence downstream of the LEH at nt 576(RHBoV1_nt576). Sequencing of a DNA fragment, detected as expected as anabout 600-bp band, confirmed the presence of the novel joint sequenceand the LEH.

The tail of the HBoV1 head-to-tail junction was found to contain asequence (5′-GCG CCT TAG TTA TAT ATA ACA T-3′; SEQ ID NO:4) identical tothat of the right-end hairpin (REH) of the other prototypic bocavirusMVC (Sun et al., 2009). Thus it was speculated that the entire HBoV1 REHis similar in structure to its MVC counterpart. Using a reverse primertargeted to this sequence (RHBoV1_nt5464) and a forward primer locatedupstream of the REH (FHBoV1_nt5201), a specific about 300-bp-long DNAfragment was amplified. Sequencing confirmed the presence of thepalindromic hairpin of the predicted REH, and revealed two novelnucleotides at the end of the hairpin.

These results indicate that the HBoV1 genome structure is typical of thegenus Bocavirus.

A Full-Length HBoV1 Clone (pIHBoV1) is Capable of Replicating andProducing Progeny Virus in HEK293 Cells

The non-structural (NS) and capsid (VP) protein-coding (NSVP) genes ofthe HBoV1 Salvador1 isolate was cloned and sequenced from thepatient-extracted viral DNA. Then the LEH, NSVP genes and REH wereligated into pBBSmaI. This sequence of the full-length genome of theisolate is deposited in GenBank (JQ923422), which is incorporated byreference herein.

It was investigated whether the adenovirus helper function is necessaryfor pIHBoV1 replication in HEK293 cells. Specifically, pIHBoV1 wastransfected into HEK293 cells (untreated or infected with adenovirus),alone or with pHelper. Interestingly, it was found that pIHBoV1replicated well in the absence of helper virus. Indeed, all the threerepresentative forms of replicated bocavirus DNA (Sun et al., 2009; Luoet al., 2011) (DpnI digestion-resistant dRF DNA, mRF DNA and ssDNA) weredetected in each test case, and at similar levels. DpnIdigestion-resistant DNA bands are newly replicated DNA in cells as DpnIdigestion only cleaves plasmid DNA prepared from prokaryotic cells,which is methylated at the dam site (Wohbe et al., 1985). In contrast,these DNA forms of the viral genome were absent in pIHBoV1-transfectedprimary airway epithelial cells (NHBE) and present at very low levels(over 20 times lower than in pIHBoV1-transfected HEK293 cells) inpIHBoV1-transfected human airway epithelial cell lines BEAS-2B, A549 and16HBE14o-, even in the presence of adenovirus. Thus, replication inthese cells appears to be non-existent or poor in these contexts.

To confirm the specificity of DNA replication and the identity of theDpnI-resistant DNA bands, the ORFs encoding viral proteins NS1, NP1, VP1and VP2 in pIHBoV1 were disrupted; knockout of expression of thecorresponding viral protein was confirmed by Western blot analysis. Whenthe NS1 ORF was disrupted, no DpnI digestion-resistant DNA was detected,confirming that replication of this DNA requires NS1. Notably, when theNP1 ORF was disrupted, an RF DNA band was detected but it was very weak,suggesting that NP1 is also involved. When the VP2 ORF was knocked out,the ssDNA band disappeared, but this was not the case when VP1 wasdisrupted (VP2 was still expressed), these findings are consistent witha role for the capsid formation in packaging of the parvoviral ssDNAgenome (Cotmore et al., 2005; Cheng et al., 2009; Plevka et al., 2011).

The presence of the ssDNA band in pIHBoV1-transfected HEK293 cellssuggested that progeny virions were produced. Large-scale pIHBoV1transfection and CsCl equilibrium centrifugation was carried out topurify the virus that was produced. The CsCl gradient was fractionated,and the highest HBoV1 gc (1-5×10⁸ gc/mL) was found at a density of 1.40mg/mL, which is typical of the parvovirus virion. Electron microscopyanalysis revealed that purified virus displayed a typical icosahedralstructure, with a diameter of about 26 nm.

Collectively, these findings confirm that a full-length clone of HBoV1capable of replicating and producing progeny virus in transfected HEK293cells was obtained.

HBoV1 Progeny Virus Produced from pIHBoV1-Transfected Cells isInfectious

The infectivity of the HBoV1 virions purified from pi HBoV1-transfectedHEK293 cells was examined in polarized primary HAE, the in vitro culturemodel known to be permissive to HBoV1 infection (Dijkman et al., 2009).Three sets (different donors, culture lots #B29-11, B31-11 and B33-11)of B-HAE were generated, and these were infected with HBoV1 from theapical side. Initially the B-HAE cultures were infected with variousamounts of virus, and when a multiplicity of infection (MOI) of about750 gc/cell was used, most of the cells (about 80%) were positive foranti-NS1 staining (indicating that the viral genome had replicated andthat genes encoded by it had been expressed) at 5 days post-infection(p.i.). This MOI was subsequently used for apical infection. Notably,B29-11, B31-11 and B33-11 HAE each supported productive HBoV1 infection.Immunofluorescence (IF) analysis of infected B31-11 HAE at 12 days p.i.showed that virtually all the cells expressed NS1 and NP1, and that agood portion of the infected cells expressed capsid proteins (VP1/2).

The production of progeny virus following HBoV1 infection was monitoreddaily by collecting samples from both the apical and basolateralchambers of the HAE culture and carrying out HBoV1-specific quantitativePCR (qPCR). In the case of B33-11 B-HAE, apical release was obviouslyinitiated at 3 days p.i., then continued to increase to a peak of about10⁸ gc/mL at 5-7 days p.i., then decreased slightly through day 10 p.i.and was maintained at a level of about 10⁷ gc/mL through day 22 p.i. Thetotal virus yield from one Millicell insert of 0.6 cm² over a 24 hourinterval was greater than 2×10¹a gc. This result suggested thatproductive HBoV1 infection of primary B-HAE is persistent. Notably, inthe B-HAE cultures from both donors, virus was also continuouslyreleased from the basolateral side, keeping pace with apical secretionthroughout, though at levels about one log lower than the release fromthe apical surface. The genomes of the progeny virions released frominfected B-HAE were amplified and sequenced. The result showed anidentical sequence with that of the HBoV1 Salvador isolate (GenbankJQ923422). Additionally, no virus was detected in mock-infected B-HAE.

Taken together, these results demonstrate that the HBoV1 virionsproduced by pIHBoV1 transfection is capable of infecting polarizedprimary HAE cultures from cells derived from various donors andreleasing identical progeny virions from infected primary HAE. Moreimportantly, we found that productive HBoV1 infection was persistent.

HBoV1 Infection of Primary B-HAE Features Characteristics ofRespiratory-Tract Injury

Although no gross cytopathic effects were observed in HBoV1-infectedB-HAE, histology analysis of mock- vs. HBoV1-infected epithelia (B33-11)revealed morphological differences: infected BHAE did not featureobvious cilia at 7 days p.i., and was significantly thinner than themock-infected one on average at 22 days p.i. The transepithelialelectrical resistance (TEER) was monitored during infection of B-HAE,and found that at 6 days it was reduced from a value of about 1,200 toabout 400 Ω·cm², while the mock-infected B-HAE maintained the initialTEER. Notably, the decrease in TEER in the infected B-HAE wasaccompanied by an increase in HBoV1 secretion.

To confirm a role for HBoV1 infection in disruption of the barrierfunction of the epithelium, the distribution of the tight junctionprotein Zona occludens-1 (Z0-1) was examined (Gonzalez-Mariscal et al.,2003). Infected B-HAE showed dissociation of ZO-1 from the periphery ofcells started from 7 days p.i., compared with mock-infected B-HAE, whichlikely plays a role in reducing TEER. Cumulatively, these resultsdemonstrate that HBoV1 infection disrupts the integrity of HAE and thatthis may involve breakdown of polarity and redistribution of the tightjunction protein ZO-1. To confirm a role for HBoV1 infection in the lossof cilia, we examined expression of the b-tubulin IV, which is a markerof cilia (Matrosovich et al., 2004; Villenave et al., 2012). InHBoV1-infected B-HAE, expression of I3-tubulin IV was drasticallydecreased at 7 days p.i., and was not detected at 22 days p.i., incontrast to that in mock-infected B-HAE. These results confirmed thatHBoV1 infection caused the loss of cilia in infected B-HAE. Notably,infected B-HAE showed changes of nuclear enlargement, which becameobvious at 22 days p.i., indicating airway epithelial cell hypertrophy.

Collectively, it was found that productive HBoV1 infection disrupted thetight junction barrier, lead to the loss of cilia and airway epithelialcell hypertrophy. These are hallmarks of respiratory tract injury when aloss of epithelial cell polarity occurs.

An Immortalized Human Airway Epithelial Cell Line Supports HBoV1Infection when the Cells are Polarized

Although primary HAE cultures support HBoV1 infection, their usefulnessis limited by the variability between donors, tissue availability andhigh cost. Alternative cell culture models were explored for theirabilities to support HBoV1 infection. Using the purified HBoV1, othercells were examined including HEK293 cells, other common epithelial celllines permissive to common respiratory viruses (Reins et al., 2001),including HeLa, MDCK, MRC-5, LLC-MK2 and Vero-E6, and severaltransformed or immortalized human airway epithelial cell lines (A549,BEAS-2B, 16HBE14o- (Cozens et al., 1994), NuLi-1 and CuFi-8 (Zabner etal., 2013), as well as primary NHBE cells for the ability to supportinfection in conventional monolayer culture. All were negative for HBoV1infection as determined by IF analysis. It was speculated that sincesome respiratory viruses infect polarized HAE but not undifferentiatedcells (Pyrc et al., 2010), some characteristics of the polarizedepithelia may be critical for HBoV1 infection. Thus immortalized cells(NuLi-1 and CuFi-8) were polarized at an air-liquid interface (ALI) forone month. Once polarization was confirmed by detection of a TEERof >500 Ω·cm², the cultures were infected with HBoV1, under the sameconditions as used for primary B-HAE cultures. Notably, IF analysisrevealed that at 10 days p.i., HBoV1-infected CuFi-HAE (differentiatedfrom CuFi-8 cells) was uniformly positive for NS1, whereas theHBoV1-infected NuLi-HAE (differentiated from NuLi-1 cells) was not.Moreover, the CuFi-HAE did express HBoV1 NS1, NP1 and VP1NP2 proteins.The kinetics of virus release from the apical surface was similar tothat of a primary B-HAE infected with virus at a similar titer(maximally 26107 gc/mL), although virus release from the basolateralsurface was undetectable. HBoV1 infection also resulted in a decrease inthe thickness of the epithelium, and dissociation of the tight junctionprotein ZO-1 from the epithelial cell peripheries.

Collectively, these findings demonstrate that the immortalized cell lineCuFi-8 (Zabner et al., 2003), when cultured and polarized at an ALI,supports HBoV1 infection, and recapitulates the infection phenotypesobserved in primary HAE, including destruction of the airway epithelialstructure.

Discussion

A full-length HBoV1 genome was cloned and its terminal hairpinsidentified. Virions produced from transfection of this clone into HEK293cells are capable of infecting polarized HAE cultures. Thus, a reversegenetics system was established that overcomes the critical barriers tostudying the molecular biology and pathogenesis of HBoV1, using an invitro culture model system of HAE.

It is notable that the HBoV1 terminal hairpins appear to be hybridrelicts of the prototype bocavirus BPV1 at the LEH, but of MVC at theREH (Schildgren et al., 2012). Replication of HBoV1 DNA in HEK293 cellsrevealed typical replicative intermediates of parvoviral DNA Althoughthe head-tail junctions are unexpected in the replication of autonomousparvoviruses, they were likely generated during the cycle of rollinghairpin-dependent DNA replication (Cotmore et al., 1987). Therefore, itis believed that the replication of HBoV1 DNA basically follows themodel of rolling hairpin-dependent DNA replication of autonomousparvoviruses, with terminal and junction resolutions at the REH and LEH,respectively (Cotmore et al., 1987). The replication of parvoviral DNAdepends on entry into S phase of the cell cycle or the presence ofhelper viruses (Cotmore et al., 1987; Berns et al., 1990). In thisregard, it is puzzling that mature, uninjured airway epithelia aremitotically quiescent (<1% of cells dividing) (Wang et al., 1999; Leighet al., 1995; Axers et al., 1988), as are the majority of the cells inpolarized HAE (in the GO phase of the cell cycle). However, recombinantadeno-associated virus (AAV; in genus Dependovirus of the family ofParvoviridae) infects HAE apically and expresses reporter genes. Geneexpression by recombinant AAV requires a conversion of the ssDNA viralgenome to a double-stranded DNA form that is capable to be transcribed.This conversion involves DNA synthesis. Hence, it was hypothesized thatHBoV1 employs a similar approach to synthesize its replicative form DNANotably, wild type AAV infected primary HAE apically and replicated whenadenovirus was co-infected. The exact mechanism of how HBoV1 replicatesin normal HAE will be an interesting topic for further investigation.

The airway epithelium, a ciliated pseudo-stratified columnar epithelium,represents the first barrier against inhaled microbes and activelyprevents the entry of respiratory pathogens. It consists of ciliatedcells, basal cells and secretory goblet cells that together with themucosal immune system, provide local defense mechanisms for themucociliary clearance of inhaled microorganisms. The polarized ciliatedprimary HAE, which is generated by growing isolated tracheobronchialepithelial cells at an ALI for on average one month, forms apseudo-stratified, mucociliary epithelium and displays morphologic andphenotypic characteristics resembling those of the in vivo humancartilaginous airway epithelium of the lung. Recent studies haverevealed that this model system recapitulates important characteristicsof interactions between respiratory viruses and their host cells.

In the current study, primary B-HAE cultures obtained from threedifferent donors were examined. HBoV1 infection of primary B-HAE waspersistent and caused morphological changes of the epithelia, i.e.,disruption of the tight barrier junctions, loss of cilia and epithelialcell hypertrophy. The loss of the former, plasma membrane structuresthat seal the perimeters of the polarized epithelial cells of themonolayer, is known to damage the cell barrier necessary to maintainvectorial secretion, absorption and transport. ZO-1, which weremonitored here, is specifically associated with the tight junctions andremains the standard marker for these structures. Similarly, cilia playimportant roles in airway epithelia, in that they drive inhaledparticles that adhere to mucus secreted by goblet cells outward. HBoV1infection compromises barrier function, and thus potentially increasespermeability of the airway epithelia to allergens and susceptibility tosecondary infections by microbes. The observed shedding of virus fromthe basolateral surface of infected primary HAE, albeit at a lower level(about 1 log lower than that from the apical surface), is consistentwith the facts that HBoV1 infection disrupted the polarity of thepseudo-stratified epithelial barrier and resulted in the leakage to thebasolateral chamber. This explanation is also supported by HBoV1infection of CuFi-HAE, where disruption of the tight junction structurewas less severe and virus was released only from the apical membrane.The induction of leakage by HBoV1 also suggests a mechanism thataccounts for the viraemia observed in HBoV1-infected patients. Furtherdisease pathology could be accounted for by infection-induced loss ofcilia of the airway epithelia; a lack of cilia is often responsible forbronchiolitis. Therefore, the data provide direct evidence that HBoV1 ispathogenic to polarized HAE, which serves as in vitro model of the lung.Since HBoV1 is frequently detected with other respiratory viruses ininfants hospitalized for acute wheezing, the apparent pathologicalchanges observed in HBoV1-infected HAE suggest that prior-infection ofHBoV1 likely facilitates the progression of co-infection-drivenpathogenesis in the patient.

The kinetics of virus release from the apical chamber of HAE infectedwith the progeny virus of pIHBoV1 (cloned from the clinical Salvador1isolate) was similar to that following infection with the HBoV1 Bonn1isolate, a clinical specimen (Dijkman et al., 2009). It is believed thatthe present study of HBoV1 infection of primary HAE reproduces infectionof the virus from clinical specimens. In addition, virus was generatedfrom a pIHBoV1-b clone, which contains the NSVP genes from the prototypeHBoV1 st2 isolate (Ailander et al., 2005). Infection of primary B-HAEwith this st2 virus resulted in a level of virus production similar tothat observed here using the Salvador1 isolate. It is believed that thestudy with the laboratory-produced HBoV1 Salvador1 represents infectionof HBoV1 of clinical specimens in HAE. The MOI used for infection in thecurrent study was high. However, it should be noted that this titer isbased on the physical numbers of virion particles as there are nopractical methods for determining the infectious titer of HBoV1preparations. It should also be taken into consideration that extensiveparvovirus inactivation occurs during the purification process, i.e.,during CsCl equilibrium ultracentrifugation (McClare et al., 2011).Virus infection of HAE most likely reflects HBoV1 infection of the lungairways in patients with a high virus load in respiratory secretions(Jartii et al., 2011).

The fact that pIHBoV1 did not replicate well in undifferentiated humanairway epithelial cells indicates that polarization and differentiationof the HAE is critical for HBoV1 DNA replication. Nevertheless,polarized NuLi-HAE, which is derived from normal human airway epithelialcells, did not support HBoV1 infection, but the CuFi-HAE derived fromairway epithelial cells isolated from a cystic fibrosis patient did. TheCuFi-HAE is unique relative to the others in that it retains thecapacity to develop epithelia that actively transport in Na+ but not Cl2because of the mutation in the cystic fibrosis gene (Zabner et al.,2003). Given the high complexity of the airway epithelium, we speculatethat the permissiveness of HBoV1 infection is dependent on various stepsof virus infection, e.g. attachment, entry, intracellular trafficking,and DNA replication of the virus. Nevertheless, a polarized CuFi-HAEmodel derived from the CuFi-8 cell line represents a novel stable cellculture model that is providing unexpected insights into the infectioncharacteristics of HBoV1. Although HBoV1 infection of CuFi-HAEreproduced disruption of the barrier tight junctions like that seen alsoin primary B-HAE, the absence of virus on the basolateral side impliesthat in HAE the secretion of HBoV1 is apically polarized. It isspeculated that the milder damage of tight junctions in these cellsmight prevent virus release from the basolateral side of infectedCuFi-HAE. Further studies will focus on understanding the permissivenessof CuFi-HAE to HBoV1 infection and on the reason for the ease ofinfection of an HAE with a cystic fibrosis phenotype.

It has been shown that HBoV1 remains detectable in the upper airways ofpatients for weeks and months, even up to half a year (Blessing et al.,2009; Martin et al., 2010; Brieu et al., 2008; Lehtoranta et al., 2010).However, the mechanism behind this persistence, i.e., whether it is dueto persistent replication and shedding, passive persistence afterprimary infection, or recurrent mucosal surface contamination, hasremained unknown. The present results in in vitro HAE cultures showedthat HBoV1 is able to replicate and shed from both the apical andbasolateral surfaces at least for three weeks, supporting the notionthat shedding of the virus from the airways is a long-lasting process.This may further explain why a high rate of co-infection, orco-detection, between HBoV1 and other respiratory viruses has beenreported. Since recombinant AAV persists as an episome in transducedtissues, which prolongs gene expression, it is possible that also theHBoV1 genome can be presented as an episome for long term expression andreplication. Apparently, the mechanism underlying this feature of HBoV1infection warrants further investigation. However, in contrast to theother human-pathogenic B19V, HBoV1 does not seem to persist in humantissues for many years (Norja et al., 2010).

In conclusion, the reverse genetics system for HBoV1 mimics naturalHBoV1 infection of the in vivo human cartilaginous airway epithelia. Thepathogenesis of HBoV1 in co-infection with other respiratory viruses andin conditions of lung diseases is a focus of future study.

Example 2

Progeny HBoV1 Virions in the Apical Washes of Infected HAE are HighlyInfectious in Polarized Primary HAE.

In Example 1, HBoV1 virions were produced from HEK293 cells transfectedwith a HBoV1 infectious clone, which were further concentrated andpurified through cesium chloride equilibrium ultracentrifugation. Duringthis process, significant viral inactivation occurred (McClure et al.,2011), and the precise infectivity of the virus was difficult todetermine. However, progeny virions were persistently secreted from theapical surface of infected HAE at a high titer (about 1.0×10⁷ vgc/μL)(Huang et al., 2012). Hence, it was hypothesized that the progenyvirions washed from the apical surface mimic naturally secreted virionsfrom HBoV1-infected lung airway-tract and thus are highly contagious.

To test this hypothesis, polarized primary HAE cultures were obtained inMillicell™ inserts of 0.6 cm² (Millipore) from the Tissue and CellCulture Core of the Center for Gene Therapy, University of Iowa. Thesecultures were made by growing isolated human airway (tracheobronchial)epithelial cells at an ALI, as described previously (Karp et al., 2002;Yan et al., 2004; Zabner et al., 1996). The inserts were kept in wellsof a 6-well tissue culture plate with 1 mL of ALI media. Infection ofthe HBoV1 progeny virions, which were collected from apical washes ofpurified HBoV1-infected primary HAE culture (Huang et al., 2012), wasanalyzed in primary HAE culture at an MOI ranged from 100 to 0.001vgc/cell from the apical surface. HBoV1 virions diluted in 150 μL of theALI media (Huang et al., 2012) were applied to the apical chanter. TheHAE cultures were incubated at 37° C. and 5% CO₂ for 2 hours. After theinoculum was removed and the apical surface was washed three times with0.4 mL of phosphate buffered saline (PBS), the cultures were returned tothe incubator. The production of progeny virions following apicalinfection was monitored daily by collecting samples from both the apicaland basolateral chambers of the HAE culture. Notably, HBoV1 virions werereleased from all these inoculated HAE cultures. However, the time topeak virus secretion was longer at lower MOIs. These times were 6, 9,12, 15, 23 and 24 days post-infection (p.i.) for MOIs of 100, 10, 1,0.1, 0.01 and 0.001 vgc/cell, respectively. Although the yields ofreleased virions at the peaks were slightly decreased along with thedecreased MOIs, a yield of about 10⁸ vgc/μL was consistently detected ininfections at MOIs from 100-0.1 vgc/cell, and a yield of about 10⁷vgc/μL was detected at MOIs of 0.01 and 0.001 vgc/cell. Mock-infectedHAE had no virus release (undetectable by quantitative PCR) from bothsurfaces. These results suggest that HBoV1 replicates in HAE slowly orpersistently.

Next, the transepithelial electrical resistance (TEER) was monitoredduring the course of infection at various MOIs. All the TEER of theinfected HAE cultures decreased drastically with an onset of decreasethat correlated with the input MOIs (at 3, 5, 7, 7, 9, 11 days p.i. forMOIs from 100, 10, 1, 0.1, 0.01 and 0.001 vgc/cell, respectively). Tosome degree, the declining curve in TEER correlated with the increasedtendency of the virus release. Nevertheless, the final TEER of all theinoculated HAE cultures by the end of the infection declined to a valueless than 400 Ω·cm², an about 2-3-fold decrease compared to that of themock-infected HAE. Destruction of the airway epithelium was alsohistologically observed. Compared with the mock-infected HAE, the degreeof airway epithelial damage at the end of infection, shown as thethickness of the epithelium and the presence of cilia, correlated withMOI of input viruses. Notably, HAE inoculated with an MOI of 100vgc/cell showed a progressive histological change. At 12 days p.i., theflattening of the HAE inoculated with an MOI of 100 vgc/cell resembledwhat was observed for HAE inoculated with MOIs of 0.1 to 0.001 vgc/cellat 26-28 days p.i. Furthermore, epithelial damage caused by HBoV1infection was substantiated by the following assays: 1) the co-detectionof HBoV1 NS1 with a significantly decreased 13-tubulin IV (a marker ofcilia (Matrosovich et al., 2004; Villenave et al., 2010)), which wasabsent in infected HAE at MOIs 100-0.1 vgc/cell and was extremely low atMOIs of 0.01 and 0.001 vgc/cell; 2) a disassociation of the tightjunction protein Zona occludens-1 (ZO-1) (11); and 3) nucleusenlargement at late infection. Early following infection, NS1 expressingcells predominantly contained little or no β-tubulin IV and haddissociated ZO-1 staining (M01=100 vgc/cell, 5 days p.i.), suggestingthat virus either initially infects non-ciliated cells or that cilia areshed early in the course of viral replication.

Collectively, it was demonstrated that the secreted progeny HBoV1virions washed from the ALI apical surface are highly infectious inpolarized primary HAE and cause severe damage of the infectedpseudostratified airway epithelia, even at an MOI as low as 0.001vgc/cell.

HBoV1 is Capable of Infecting Polarized Primary HAE from the BasolateralSurface.

HBoV1 apical infection of primary HAE was persistent and that progenyvirions were secreted from both the apical and basolateral surfaces(Example 1). However, whether HBoV1 infects primary HAE from thebasolateral surface remains elusive. To address this question, naiveprimary HAE were inoculated basolaterally with apically washed HBoV1virions, as used above at an MOI of 1 vgc/cell.

For basolateral infection, HBoV1 virions were diluted in 1 mL of the ALImedia in the basolateral chamber of HAE cultures. The cultures wereincubated at 37° C. and 5% CO₂ for 2 hours. Then the basolateralinoculums were removed and washed twice with 1 mL PBS. After addition offresh media, the cultures were returned to the incubator. Production ofprogeny virions following basolateral inoculation was monitored daily bycollecting samples from both the apical and basolateral chambers.Following basolateral inoculation, the apical viral secretion increasedslowly, but to a peak of about 5×10⁷ vgc/μL at 16 days p.i. Virionrelease was maintained at a level of over 10⁶ vgc/μL throughout thecourse of infection. Progeny virions were also released from thebasolateral surface following the basolateral inoculation, but at alevel of about 1 log less than that from the apical surface over thecourse of infection, which is similar to what was observed followingapical infection. This result suggests that HBoV1 infection of HAE fromthe basolateral surface is also persistently productive, similar to whatwas observed following apical infection.

TEER was also monitored during basolateral infection. While the TEER ofthe mock-infected HAE cultures was consistently at a level of around1000 Ω·cm² during the experiment period, the TEER ofbasolaterally-inoculated HAE dropped gradually and to a value of about400 Ω·cm² at 15 days p.i., seen slightly earlier (13 days p.i.) inapically-inoculated HAE (M01=1). This result is consistent with theviral release kinetics, indicating that HBoV1 infection disrupts theepithelial barrier. Basolaterally-inoculated HAE showed a cleardissociation of the tight junction, compared with that in themock-infected HAE.

By the end of infection, at 22 days p.i., a histology analysis ofbasolaterally-inoculated HAE was performed. In contrast to the mockcontrol, infected HAE showed an absence of cilia and an obviouslythinner epithelium. This observation was further confirmed by theabsence of β-tubulin IV expression. The infected HAE also showed nuclearenlargement at late-stage infection (DAPI).

Taken together, these results demonstrate that the HBoV1 is capable ofinfecting polarized primary HAE from the basolateral surface. They alsoshow that the basolateral infection is persistently productive, causesloss of the cilia, and ultimately disrupts the tight junction barrier ofthe epithelium. However, in comparison to apical HBoV1 infection,basolateral infection is less efficient, suggesting that HBoV1 infectionhas a stronger apical tropism. The basolateral infection suggests thatHBoV1 viremia (Kantola et al., 2008; Nascimento-Carvalho et al., 2012)may facilitate viral infection all over the lung airway tracts inpatients.

Overall, this study demonstrates that HBoV1 productively infectspolarized primary HAE at a low MOI both at the apical and basolateralsurfaces. Mature and uninjured airway epithelia are mitoticallyquiescent (<1% of cells dividing) (Ayers et al., 1988; Lergh et al.,1995; Wang et al., 1999).

Example 3

Materials and Methods

Plasmids.

pIHBoV1 is the infectious clone plasmid containing the 5543 bpfull-length HBoV1 genome (Huang et al., 2012). prHBoV1-CBAluc is arecombinant HBoV1 (rHBoV1) transfer plasmid derived from pIHBoV1 and wasconstructed by replacing the 2.64 kb HindIII/BglII fragment of the HBoV1genome by a 2.74 kb fragment containing the CMV enhancer/chicken β-actinpromoter driven Luciferase gene. The NP1 gene, which plays an essentialrole in HBoV1 DNA replication, was completely removed in the resultingprHBoV1-CBAluc plasmid. To reduce the probability of rescued wild typevirus through recombination, the 5′ remained NS gene coding region wasfurther disrupted by elimination of a BspE1 site using blunt ligation,and the 3′ VP partial coding region was further deleted by removal of a145 bp PstI to EcoRI fragment. The helper plasmid pHBoV1KUm630 harborsthe 5299 bp HBoV1 genome (99 to 5395-nt) without terminal repeats (Chenet al., 2010), with the P5 promoter and 3′ polyA signal being retainedfor the expression of viral genes. pAV-Rep2 and pAD4.1 are the helperplasmids supporting rAAV2 genome rescue and replication from proviralplasmids in 293 cells as described previously. pAV2-F5tg83luc is a rAAV2cis transfer plasmid, containing a 4.85 kb rAAV proviral genome with a180 bp synthetic promoter driving firefly luciferase gene.pAV2-CF5tg83luc is a longer form of pAV2-F5tg83luc, and was derived byinserting 600 bp of stuffer sequence upstream the 180 bp syntheticpromoter to generate a rAAV2 proviral genome 5.4 kb in length.pAV2-CBAhCFTR harbors an oversized 5.5 kb rAAV2 proviral genomecontaining a human CFTR expression cassette with a 580 bp CMV IEenhancer plus β-actin promoter (CBA promoter), a 50 bp synthetic polyAsignal, and a 4443 bp human CFTR cDNA containing 56 bp 5′UTR and 45 bp3′UTR.

Recombinant Virus Production.

rAAV vectors, rAAV2/2.F5tg83luc and AV2/1.F5tg83luc, were generated bytriple plasmid co-transfection using an adenovirus-free system in HEK293cells as described in Yan et al. (2006); this system uses the rAAV transhelper plasmid pAVRC2.3, adenovirus helper plasmid pAD Helper 4.1, andrAAV2 proviral plasmid pAV2-F5tg83luc, transfected at a ratio of 2:3:1,respectively. The rHBoV1 vector stock (HBc.CBAluc) was generated byco-transfection of helper pHBoV1KUm630 and proviral plasmidprHBoV1-CBAluc into HEK293 cell at a ratio of 3:1, respectively.Chimeric rAAV2/HBoV1 vectors were generated by pseudotyping the rAAVgenome within HBoV1 capsid following co-transfection of pAV-Rep2,pAd4.1, pHBoV1KUm630 together with the rAAV cis proviral plasmid intoHEK293 cells at a ratio of 1.5:3:3:1, respectively. The rAAV proviralplasmids used for the chimeric vector production were pAV2-F5tg83luc(4.8 kb), pAV2-CF5tg83luc (5.4 kb) and pAV2-CBAhCFTR (5.5 kb). Allviruses were recovered from the cell pellets at 72 hourspost-transfection and the cell crude lysates were treated as for rAAVvector production as described in Yan et al. (2013). After DNase Idigestion, all viruses were purified by two rounds of CsClultracentrifugation and dialyzed against PBS. The titers of viralpreparations as DNase I-resistant particles (DRP) were determined byTaqMan real time quantification PCR and confirmed with slot blot assaysusing a ³²P-labeled probe against the luciferase gene (Yan et al.,2013).

Western Blotting.

5×10⁹ DRP of rAAV2 and chimeric rAAV2/HBoV1 were resolved by 10%SDS-PAGE. Following transfer to nitrocellulose membranes, two-colorWesterns were performed with mouse anti-MV capsid monoclonal antibody B1(1:1000) and rat anti-HBoV1 VP2 antiserum (recognizing both VP1 and VP2proteins) (Chen et al., 2010) (1:200). Infrared detection used 1:10,000dilution of the secondary antibody goat anti-mouse-IRDye700 (red, forAAV) and goat anti-rat-IRDye800 (green, for HBoV1). Images were thenscanned using an Odyssey Infrared Image System.

Cell Culture and Virus Infection Conditions.

HEK293 and IB3 cells were cultured as monolayers in Dulbecco's modifiedEagle medium (DMEM), supplemented with 10% fetal bovine serum andpenicillin-streptomycin, and maintained in a 37° C. incubator at 5% CO₂.Undifferentiated immortalized CF human airway cells (CuFi8) werecultured as monolayers in bronchial epithelial cell growth medium (BEGM,Lonza) (Zabner et al., 2003). Polarized primary human airway epitheliawere generated as previously described from lung transplant airwaytissue (Yan et al., 2004; Karp et al., 2002) and were obtained from theTissue and Cell Culture Core of The Center for Gene Therapy at theUniversity of Iowa. Epithelia were grown on 12 mm Millicell membraneinserts (Millipore) and differentiated with 2% USG medium at anair-liquid interface prior to use. Polarization of CuFi8 cells at an ALIwas performed using similar conditions to primary HAE. To apicallyinfect the polarized airway epithelia, 1×10¹⁰ DRP of virus was dilutedin USG medium to the final volume of 50 it and applied to the upperchamber of the Millicell insert. For basolateral infections, 1×10¹⁰ DRPof virus was directly added to the culture medium in the bottom chamber.Viruses were typically exposed to epithelia for 16 hrs and then removed.At this time, the Millicell inserts were briefly washed with a smallamount USG medium and fed with fresh USG medium in the bottom chamberonly. Approximately 1-2×10⁶ cells are in each Millicell insert and thusthe multiplicity of infection (MOI) is estimated about 5,000 to 10,000DRP/cell. Transduction was assessed by luciferase reporter assays atvarious time points post-infection using cell lysates or IVISbiophotonic imaging.

Measurement of Luciferase Reporter Expression.

Luciferase enzyme activity in cell lysates was determined using theLuciferase Assay System (Promega) in a 20/20 luminometer equipped withan automatic injector (Turner Biosystems). Quantification of luciferaseactivity in live cells was performed using the IVIS Biophotonic Imagingsystem according to the manufacturer's instructions. Images werecaptured 15 minutes after adding the VivoGlo Luciferin substrate(Promega) to the basolateral culture medium only, and quantification ofimages were processed with the Living Image 2.51 software (Xenogen).

Analysis of Internalized Viral Genome.

Fully-differentiated human polarized airway epithelia were infected with1×10¹⁰ particles of rAAV or chimeric rAAV/HBoV1 vectors. After a 4 hrinfection period, virus was removed, and epithelia were extensivelywashed with PBS. The Millicell inserts then were fed with fresh mediumin the bottom chamber and placed in a 37° C. incubator for 18 hours.Prior to harvesting cells for all viral internalization assays,Millicell inserts were washed thoroughly with 40 mL PBS in a 50 mLconical tube three times. The cells were then detached from the supportmembrane of the Millicell inserts by trypsin digestion. The cell pelletswere then washed three more times with 1 mL PBS prior to subcellularfractionation and viral genome quantification. Control experimentsutilizing virus bound for 1 hour at 4° C. demonstrated >98% removal ofvirus from the cell surface using this washing and trypsin digestionmethod (data not shown). Nuclei were isolated with the Nuclei EZ pre kit(Sigma) as described in Chen et al. (2011). The cytoplasmic fractionswere pooled during the nuclei preparation and 1/10 was dried in aSpeedVac. The nuclei pellet and dried cytoplasmic fraction weredissolved in 50 μL digestion buffer (50 mM KCl, 2.5 mM MgCl₂, 10 mM TrispH 8.0, 0.5% NP40, 0.5% Tween-20 and 400 μg/ml proteinase K). Afterdigestion at 56° C. for 45 minutes and heat-inactivation at 95° C. for15 minutes, 0.1 μL of the nuclear and 1 μL of the cytoplasmic digestionwere used for TaqMan PCR to quantify viral genomes. When total viralinternalization assays were performed in CuFi ALI cultures,non-polarized CuFi and HEK293 cell monolayers, the same washing andtrypsinization procedure was used to remove cell-surface bound virions.However, the washed cell pellets were directly lysed with the abovedigestion buffer and used for viral genome quantification.

Quantitative Analysis of rAAV Genomes by TaqMan PCR.

TaqMan real time PCR was used to quantify the physical titer of theviral stocks and copies of viral genomes in cell lysates from AAVinfected cells as described in Yan et al. (2006). The PCR primers usedwere 5′-TTTTTGAAGCGAAGGTTGTGG-3′ (SEQ ID NO:6) (forward) and5′-CACACACAGTTCGCCTCTTTG-3′ (SEQ ID NO:7) (reverse) and amplify a 73 bpfragment of the rAAV2.Luc genome. The Taqman probe(5′-ATCTGGATACCGGGAAAACGCTGGGCGTTAAT-3′) (SEQ ID NO:8) was synthesizedby IDT (Coralville, Iowa). This probe was tagged with 6-carboxyfluorescein (FAM) at the 5′-end as the reporter and Dark Hole Quencher 1(BHQ1) at the 3′-end as the quencher. The PCR reaction was performed andanalyzed using Bio-Rad MyIQ™ Real-time PCR detection system andsoftware.

Short Circuit Current Measurements.

Transepithelial short circuit currents (Isc) were measured using anepithelial voltage clamp (Model EC-825) and a self-contained Ussingchamber system (both purchased from Warner Instruments, Inc., Hamden,Conn.) as described in Lin et al. (2007). Throughout the experiment thechamber was kept at 37° C., and the chamber solution was aerated. Thebasolateral side of the chamber was filled with buffered Ringer'ssolution containing 135 mM NaCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 2.4 mMKH₂PO₄, 0.2 mM K₂HPO₄, and 5 mM Hepes, pH 7.4. The apical side of thechamber was filled with a low chloride Ringer's solution in which 135 mMNa-gluconate was substituted for NaCl. Transepithelial voltage wasclamped at zero with current pulses every 5 seconds to record theshort-circuit current using a VCC MC8 multichannel voltage/current clamp(Physiologic Instruments) with Quick DataAcq software. The followingchemicals were sequentially added into the apical chamber: (1) amiloride(100 μM) for inhibition of epithelial sodium conductance by ENaC; (2)4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS) (100 μM) toinhibit non-CFTR chloride channels; (3) cAMP agonists forskolin (10μM)/3-isobutyl-I-methylxanthine (IBMX) (100 μM) to activate CFTRchloride channels; and (4) 10 μM CFTRinh-GlyH-101(N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl) methylene]glycine hydrazide) to block Cl⁻ secretion through CFTR. Δlsccalculations were made by taking the difference of the plateaumeasurements average over 45 seconds before and after each experimentconditions (chemical stimulus).

Cftr Immunostaining.

Following short circuit current measurements, the HAE on the supportmembrane was cut out from the Millicell insert and embedded in OCTmedium. 10 μm cryosections were fixed in 4% paraformaldehyde followed byblocking and immunofluorescence staining using an anti-CFTR antibodycocktail consisting of equal amount (at a 1:200 dilution) of three mouseanti-CFTR antibodies (clone MM13-4 (Millipore), clone M3A7 (Millipore),and clone 131 (R&D system)) and finally incubation with donkeyanti-mouse FITC-labeled secondary antibody.

Results

Production of a Recombination HBoV1 (rHBoV1) Vector and its TransductionProperties in HAE Model Systems.

The plasmid clone of the full length HBoV1 genome (pIHBoV1) can be usedto produce infectious HBoV1 virions following transfection in HEK293cells without the need for helper virus functions (Huang et al., 2012).It was first attempted to generate rHBoV1 by testing if HBoV1 viralproteins could trans-complement and rescue replication and packaging ofa rHBoV1 genome in HEK293 cells. The structure of the wild type HBoV1genome found in the infectious plasmid (pIHBoV1), the rHBoV1 proviralplasmid (prHBoV1-CBAluc), and the trans-helper plasmid (pHBoV1KUm630)are schematically shown in FIG. 1A. The prHBoV1-CBAluc contained a 5.5kb rHBoV1 proviral genome encoding a CBA promoter-driven luciferasegene. The ability of pHBoV1KUm630 to support rHBoV1 genome rescue andreplication from prHBoV1-CBAluc was confirmed by co-transfecting theseplasmids into HEK293 cells. Hirt DNA extracted from transfected cellswas evaluated by Southern blot following DpnI digestion to eliminate themethylated plasmid background signal. As shown in FIG. 1B, rHBoV1replication intermediates were only observed in cells co-transfectedwith prHBoV1-CBALuc and pHBoV1KUm630, but not with prHBoV1-CBALuc alone.Although the detected replication intermediates from the co-transfectionwere lower than the level from the wild type HBoV1 proviral plasmidpIHBoV1, we conclude the pHBoV1KUm630 plasmid does provide the necessaryhelper functions in trans to support rHBoV1 genome replication (FIG.1C). To generate recombinant virus, prHBoV1-CBAluc and pHBoV1KUm630 wereco-transfected into HEK293 followed by purification from cell lysateswith CsCl equilibrium ultracentrifugation. Fractions from this gradientwere evaluated for viral genomes by TaqMan PCR and demonstrated a peakat a density of 1.45 to 1.40 g/mL (FIG. 1C), suggesting successfulencapsidation of viral DNA. Viral yields from thirty 150-mm platesyielded a total of 1.25×10¹¹ rHBoV1 genome copies (vc) or DNase Iresistant particles (DRP). This is roughly 20% of the yield of wild typeHBoV1 obtained from the transfection of pIHBoV1 in HEK293 cells (about2×10¹¹ DRP per ten 150 mm dishes) (Huang et al., 2012).

Twice-CsCl banded rHBoV1.CBAluc was then evaluated for its transductionproperties following infection of HEK 293 cells, 163 cells (a CF humanairway cell line), monolayers of CuFi8 cells (a conditional transformedCF airway cell line (Zabner et al., 2003)), and ALI cultures derivedfrom CuFi cells and primary human airway epithelial cells (FIG. 1D).Results demonstrated that rHBoV1.CBAluc was only capable of transducing(i.e., expressing its encoded transgene) polarized and differentiatedALI cultures derived from either primary or CuFi airway cells. Thesefindings are similar to conditions required for productive infectionwith wild type HBoV1 (Huang et al., 2012). Following apical infection ofpolarized HAE with rHBoV1.CBAluc, maximal transgene expression occurredat 3 days post-infection and gradually declined by 11 dayspost-infection (FIG. 1E).

Encapsidation of a Recombinant AAV2 Genome in HBoV1 Virions ThroughCross-Genera Parvovirus Pseudotyping.

Although wild type HBoV1 and rHBoV1 virions can be assembled in HEK293cells following plasmid(s) transfection, the viral yields are relativelylow. By contrast, rAAV vector replication is very efficient in HEK293with the proper helper plasmids. We reasoned that HEK293 cell machinerysupporting HBoV1 replication could be less efficient because the HEK293cells are not a biologically permissive cell line for HBoV1 productiveinfection. Therefore, it was explored whether pseudotyping rAAV2 genomeswith HBoV1 capsids would generate a rAAV2/HBoV1 chimeric vector withhigher yields in these cells. Using this approach, the 4.85 kb rAAVgenome from the cis rAAV proviral plasmid (pAV2-F5tg83luc) wassuccessfully packaged into HBoV1 virions following co-transfection withpAD4.1 (encoding all the necessary helper function for AAV replicationfrom adenovirus), pAV-Rep2 (encoding the AAV2 Rep genes), andpHBoV1KUm630 (encoding the HBoV1 capsids and NS genes). There wereseveral reasons for using a helper plasmid that expressed all HBoV1viral proteins to support encapsidation of the rAAV2 genome. First, thetemporal regulation and transcriptional profiles of HBoV1 genes requiredto support packaging remain unknown. Second, the functional roles ofHBoV1 NS and NP1 proteins in capsid assembly are unclear. Lastly, it ispossible that NS proteins associate with AAV2 ITRs and this couldpotentially help to facilitate packaging of AAV2 genomes into HBoV1capsids. Three days after co-transfection, substantial DNase I-resistantviral particles were recovered from crude lysates by CsCl banding (FIG.2A). Omitting pAV-Rep2 from the transfection cocktail failed to produceDNase I resistant viral particles. Of note, the density of the chimericrAAV2/HBoV1 virions was about 1.435 g/mL, similar to that of the rHBoV1virions (FIG. 1A). This density was slightly heavier than the 1.414 g/mldensity of the rAAV2/2 virions (FIG. 1A). The typical yields of thechimeric rAAV/HBoV1 vector were 10- to 20-fold less than rAAV2 vector,but 2- to 4-fold greater than rHBoV1 vector, when generated on a similarscale. By contrast, the pseudopackaging of a rAAV genome into humanparvovirus B19 capsid is much less efficient than what we observed forrAAV2/HBoV1 (Ponnazhagan et al., 1998).

Examination of rAAV2 genome rescue and replication demonstrated thatrAAV2 replication from DNA (RF DNA) in the rAAV2/HBoV1 production systemwas about 2 to 3-fold less abundant than that during rAAV2 production(FIG. 2B, compare lanes 2 and 4). This is consistent with previousreports describing that the parvovirus capsid plays a feedback role invirion formation (Cheng et al., 2010; Huang et al., 2012). Supportingthis, it was observed that less rAAV2 RF DNA was present followingco-transfection of the AAV2 proviral plasmids (pAV2.F5tg83luc andpAV2.CF5tg83luc) and the pAV2-Rep helper (missing intact AAV capsidgenes) (FIG. 2B, lane 1 and lane 3), than following co-transfection ofpAV2.F5tg83luc and the AAV2 Rep-Cap helper plasmid pAVRC2.3 (FIG. 2B,lane 2). Of note, the expression of the HBoV1 viral proteins appeared tonot interfere with rAAV2 genome rescue and replication (FIG. 2B, comparelane 4 vs. lane 1 or 3). Transmission electron microscopy (EM)demonstrated that rAAV2/HBoV1 virions had a typical parvovirusicosahedral structure that was 26 nM in diameter (FIG. 2C), similar towild type HBoV1 virions (Huang et al., 2012). The density of theinterior of the virions by EM also suggested that >99% of virions werefully packaged with DNA. Examination of the chimeric rAAV2/HBoV1 virionsby Western blot with anti-HBoV1 VP2 antiserum demonstrated the presenceof HBoV1 VP1 and VP2 proteins, but no AAV2 VP proteins were detected inthe rAAV/HBoV1 stock using an anti-AAV2 capsid monoclonal antibody B1(FIG. 2D).

Characterization of Viral Genome Polarity and Capacity of rAAV2/HBoV1Virions.

One significant difference between wild type AAV2 and HBoV1 virions isthe polarity of packaged genomes—about 50% of AAV virions contain a plusDNA strand, while the other half contain a minus DNA strand, whereasHBoV1 selectively encapsidates the minus DNA strand more than 90% of thetime (Schildgren et al., 2012). The two terminal palindromic sequencesof HBoV1 are asymmetric (differing in size, primary sequence, andpredicted structure (Huang et al., 2012)), while for MV, terminalpalindromic sequences are identical inverted repeats. The terminalsequences in parvovirus genomes are critical to the formation ofconcatameric duplex replication intermediates and excision of singlestranded progeny genomes for packaging (Cotmore et al., 1996). Giventhat the rAAV2/HBoV1 vector genome has identical inverted repeats at theends of its genomes, it was hypothesized that rAAV2/1-1BoV1 would adoptunbiased packaging of both the plus and minus strands. This was indeedthe case. Using sense and antisense probes against the luciferasetransgene, rAAV2/HBoV1 virion DNA demonstrated approximately equalproportions of plus and minus strands, while rHBoV1 vector DNAdemonstrated a preference (about 87%) for packaging the minus strand.These differences may, in part, account for the 2-3 fold higher yield ingeneration of rAAV2/HBoV1 over that of rHBoV1.

A second major difference between MV and HBoV1 genomes are theirsize—the AAV genome is 4679-nt in length, while the HBoV1 is 5543-nt inlength. The packaging capacity of rAAV vectors has been extensivelystudied and has limits of 4.9 to 5.0 kb (Dong et al., 1996; Wa et al.,2010). This is a significant hurdle for delivery the CFTR gene by rAAV,and one that might be potentially overcome with rAAV2/HBoV1 vectors.Thus, it was hypothesized that the rAAV2/HBoV1 particle might offer asignificant advantage for CFTR delivery by virtue of its ability topackage oversized rAAV genomes up to 5.5 kb, as observed with rHBoV1(FIG. 1D). To explore this possibility, two rAAV proviral genomes weregenerated with identical luciferase expression cassettes that differedin length by 600 bp (pAV2/HBc.F5tg83Luc at 4.8 kb andpAV2/HBc.CF5tg83Luc at 5.4 kb). Each of these proviral plasmids was usedto generate rAAV2/2 (4.8 kb) and/or rAAV2/HBoV1 (4.8 and 5.4 kb)viruses, and the viral DNA was evaluated by alkaline-denatured agarosegel electrophoresis followed by Southern blot analysis. The viral yieldsof 5.4 kb rAAV2/HBoV1 was similar to 4.8 kb rAAV2/HBoV1. The Southernblot analysis of viral DNA revealed only genomes of the appropriate sizewith no obvious truncated forms (FIG. 3B). These findings demonstratethat HBoV1 pseudotyping accurately processes and packages both short andlong rAAV genomes without altering genome integrity.

Viral genome recombination plays an important role in the evolution ofmany viruses, and strand recombination also occurs during thereplication of single-stranded viruses (Martin et al., 2011). Enterichuman bocavirus infections are also associated with a high level ofviral genome recombination (Kapoor et al., 2010). It was evaluatedwhether recombination products between the helper and proviral plasmidswere packaged into rHBoV1 and rAAV2/HBoV1 virions. Slot blot analyses ofviral genomes from purified rAAV2/HBoV1 and rHBoV1 were conducted usinga luciferase transgene probe and HBoV1 helper-specific viral probe(i.e., the HindIII/BglII 2.64 kb fragment replaced by the luciferasecassette in the rHBoV1 vector). Results from this analysis demonstratedthat 17% of the viral genomes in purified rHBoV1 stocks contained HBoV1sequences found only in the helper plasmid (FIG. 3C). The inclusion of1.4 kb and 1.1 kb HBoV1 genome fragments flanking the luciferasecassette in the rHBoV1.CBAluc genome is the mostly likely cause of theserecombination events. Although both the NS and VP protein coding domainswere mutated in the rHBoV1.CBAluc genome (FIG. 1A), if recombinationoccurred outside these mutations in a double cross-over event,replication-competent rHBoV1 genomes would be expected in rHBoV1 viralstocks. The presence of replication-competent virus could be one of thereasons for the time-dependent decline in transgene expression of rHBoV1infected HAE ALI cultures (FIG. 1E). In contrast to rHBoV1. HBoV1 helpergenomes were not detected in purified rAAV2/HBoV1 virus, as would beexpected, since there is no sequence homology between the proviral andhelper plasmids. Thus, further development of packaging strategies forrHBoV1 are needed to eliminate the chance of generating replicationcompetent HBoV1 (i.e, expression of NS and VP genes on separate helperplasmids and minimal cis-elements for packaging in the proviral genome).However, improved knowledge of the regulation of NS/VP viral genes andHBoV1 genome packaging will be needed before similar strategies thateliminate replication competent virus in the generation of rAAV can beapplied.

Chimeric rAAV2/HBoV1 Vectors Mediate Highly Efficient Transduction fromthe Apical, but not Basolateral Membrane, of Human Polarized AirwayEpithelia.

Next the transduction characteristics of the rAAV2/HBoV1 chimericvectors was examined. A rAAV2/HBoV1 vector encoding the luciferasetransgene failed to transduce HEK293 cells at even high MOIs (50,000DRP/cell), while the analogous rAAV2/2 vector efficiently expressedluciferase at much lower MOIs (2,500 DRP/cell) (FIG. 4A). Experiments inprimary HAE and CuFi ALI cultures confirmed that both AV2/HBc.F5tg83luc(4.8 kb genome) and AV2/HBc.CF5tg83luc (5.4 kb genome) gave rise tosimilar levels of luciferase expression following apical infection (datanot shown), suggesting that vector size within this range did not impacttransduction. Next the transduction of rAAV2/HBoV1 was compared to thatof rAAV vectors under the same infection conditions. This directcomparison was only possible with AV2/HBc.F5tg83luc, AV2/1.F5tg83luc andAV2/2.F5tg83luc (with identical proviral genomes derived frompAV2-F5tg83luc), since the pAV2-CF5tg83luc genome (5.4 kb) was too largeto be packaged into AAV capsids. The transduction patterns for primaryHAE following apical and basolateral infection with AV2/HBc.F5tg83lucand AV2/2.F5tg83luc were strikingly different (FIG. 4B). As previouslyobserved, rAAV2/2 transduced HAE with a strong basolateral preference(Yan et al., 2006: Yan et al., 2004) the luciferase expression followingapical infection was 210-fold lower than that following basolateralinfection (FIG. 4B). By contrast, rAAV2/HBoV1 demonstrated a 206-foldgreater level of transduction following apical infection of primary HAEas compared to basolateral infection (FIG. 4B). Importantly, the levelof transgene expression achieved following apical infection withAV2/HBc.F5tg83luc was 70-fold greater than that from AV2/2.F5tg83luc,and 5.6-fold greater than AV2/1.F5tg83luc. As previously demonstrated,rAAV2/1 lacked a polarity bias for transduction in polarized HAE, andhad better apical transduction efficiency than rAAV2 (Yan et al., 2006).

Since HBoV1 is a recently discovered virus, little is known aboutHBoV1-cell interactions in HAE. Wild type HBoV1 can infect HAE at MOIsas low as 0.001 DRP/cell (Deng et al., 2013), suggesting that viralentry from the apical surface of HAE is quite efficient. However, withthe added variable of viral replication, the efficiency of HBoV1internalization and intracellular trafficking to the nucleus isdifficult to directly evaluate. The creation of a replication defectiverAAV2/HBoV1 chimeric virus provides the opportunity to directly evaluatethese processes and furthermore compare the efficiency of these steps intransduction with rAAV vectors. To this end, virion uptake and viralgenome distribution was compared in primary ALI cultures of HAE at 18hours following apical infection. As controls, we included two rAAVpseudotyped vectors, rAAV2/1 (AV2/1.CMVluc) and rAAV2/2 (AV2/2.CMVluc),which are known to transduce HAE from the apical membrane with differentefficiencies (Yan et al., 2006). rAAV1 has thus far been shown to be oneof the most efficient AAV serotype for transduction of HAE, with greatervirion uptake and faster nuclear translocation following apicalinfection (Yan et al., 2013; Yan et al., 2006). Additionally, bothAV2/HBc.F5tg83luc and AV2/2.F5tg83luc viruses that contain the identicalviral genome were evaluated. Results from these comparisons demonstratedthat AV2/1.CMVluc and AV2/HBc.F5tg83luc showed about 14-fold and about32-fold more viral uptake from the apical membrane of primary HAE thanthe rAAV2/2 vectors (AV2/2.CMVluc and AV2/2.F5tg83luc), respectively(FIG. 4C). AV2/HBc.F5tg83luc viral uptake was also 2.3-fold moreefficient than AV2/1.CMVluc (P=0.026). Furthermore, the post-entryprocessing of rAAV2/HBoV1 to the nucleus appeared to be more rapid thanfor rAAV2/2, with about 15% of internalized AV2/HBc.F5tg83luc genomesdetected in the nuclear fraction by 18 hours post-infection, as comparedto about 7% for rAAV2/2 (FIGS. 4C and D). Interestingly, thecytoplasmic/nuclear distribution of viral genomes was similar forrAAV2/1 (86.4/13.6%) and rAAV2/HBoV1 (85.0/15.0%) (FIGS. 4C and D). Incontrast to both rAAV serotypes tested, rAAV2/HBoV1 poorly transducedHAE from the basolateral membrane (FIG. 4B). Overall, these findingssuggest that rAAV2/HBoV1 viral uptake and nuclear translocation ishighly efficient following apical infection of primary differentiatedHAE.

Modulating Proteasome Activity During the Infection Period GreatlyEnhances Transduction Following Apical Infection with rAAV2/HBoV1.

Despite the fact that rAAV2/HBoV1 demonstrates a high transductionefficiency following apical infection in HAE ALI, the majority (85%) ofinternalized rAAV2/HBoV1 virions are retained in the cytoplasm at 18hours post-infection. This suggested that intracellular barrierslimiting effective nuclear transport of the virus, as observed for rAAV2and rAAV1 transduction of HAE (Duan et al., 2000; Duan et al., 1998),may also exist for HBoV1. Thus, the transduction efficiency ofrAAV2/HBoV1 could be further improved by overcoming these barriers.Impaired endosomal processing/intracellular trafficking is one of themajor barriers that limit rAAV vector transduction of polarized HAEfollowing apical infection. This barrier can be partially overcome bythe application of proteasome inhibitors at the time of infection orwithin a certain period after infection (Duan et al., 2000; Zhang etal., 2008; Yan et al., 2004). These studies have demonstrated that bothtripeptidyl aldehyde N-acetyl-1-leucyl-1-leucyl-1-norleucine (LLnL) andthe anthracycline derivative doxorubicin (Dox) can enhance the rAAV2,rAAV5, and rAAV1 viral processing and translocation to the nucleus,leading to higher levels of transduction. Combined administration ofthese two distinct classes of proteasome activity modulating agents caninduce transduction over 1000-fold following apical rAAV2/2 infection ofprimary HAE (Yan et al., 2006; Yan et al., 2004). Although there is asignificant divergence between the HBoV1 and AAV2 capsid proteins atprimary sequence level, these two viruses retain some conserved corecapsid sequences and also share a similar surface icosahedral topologywith other parvovirus particles (Gurda et al., 2010). Thus, it washypothesized that treatment with proteasome inhibitors might alsoenhance transduction of HAE by rAAV2/HBoV1, and sought to study thekinetics of transduction between rAAV2/2 and rAAV2/HBoV1 in the presenceand absence of LLnL and Dox. Results comparing rAAV2/2 to rAAV2/HBoV1(FIGS. 5A and B) demonstrated a similar rise in transgene expressionbetween 3-7 days post-infection, with a plateau at 7-11 dayspost-infection. For both vectors, treatment with proteasome inhibitorsat the time of infection enhanced transduction at all time pointsgreater than 1000-fold, and there was no decline in transgene expressionat the 11 day period. These findings demonstrate that rAAV2/HBoV1 sharesa similar proteasome-dependent barrier to transduction as observed withmost other rAAV serotypes.

Polarization of Human Airway Epithelia is Required for HBoV1Capsid-Mediated Gene Transfer.

The mechanism by which HBoV1 productively infects the apical membrane ofpolarized human airway epithelia remains unclear. In previous studies,we observed that infection of monolayers of CuFi cells with wild-typeHBoV1 does not support viral replication and production of progenyvirions (Huang et al., 2012), a finding similar to the lack oftransduction of monolayer CuFi cells with luciferase expressing rHBoV1(FIG. 1D). By contrast, when polarized, CuFi cells efficiently produceprogeny virus following apical, but not basolateral, infection withwild-type HBoV1 (Huang et al., 2012). These findings suggest thatpolarization influences a cellular factor(s) required for productiveinfection, such as expression of a viral receptor/co-receptor orexpression of factors involved in intracellular processing of the HBoV1virion. Since viral replication does not occur within the chimericrAAV2/HBoV1 vector, this was an opportunity to define the step(s)following HBoV1 infection that are influenced by polarization. To thisend, experiments were performed with rAAV2/HBoV1 to address whether thepolarization of airway epithelial cells influences HBoV1 capsid-mediatedtransduction through steps involving receptor binding/uptake or thepost-entry intracellular processing of virions. Since theubiquitin-proteasome pathway affects rAAV2/HBoV1 transduction in HAE ALIcultures, we also examined the influences of proteasome inhibitors onthese two steps of infection.

Equivalent numbers of CuFi cells under monolayer (i.e., non-polarized)or polarized ALI culture conditions were incubated with equal amounts ofAV2/HBc.F5tg83luc virus at 37° C. for 4 hours. After removal of theunbound vectors, the infected cells were either lysed for quantificationof internalized vector genomes by TagMan PCR or cultured for anadditional 20 hours prior to assessing transgene expression (FIG. 5C).Results from this analysis demonstrated that apical transduction ofpolarized CuFi ALI cultures with AV2/HBc.F5tg83luc was 72-fold moreefficient than basolateral transduction, a finding consistent withAV2/HBc.F5tg83luc infection of primary HAE ALI cultures (FIG. 4B). Underthese conditions, about 40-fold more virus was taken up by CuFiepithelia following a 4 hours apical infection as compared tobasolateral infection (FIG. 5C), suggesting that polarization enhancesthe abundance of HBoV1 receptor/co-receptor on the apical membrane.Interestingly, AV2/HBc.F5tg83luc entered CuFi cell monolayers at anefficiency similar to that observed following apical infection ofpolarized CuFi epithelia (FIG. 5C), despite significantly reducedtransduction of non-polarized CuFi cultures (FIG. 4B). Additionally,analysis of viral uptake and transduction of HEK293 cells infected withAV2/HBc.F5tg83luc, which is not permissive to HBoV1 infection, revealedsubstantial viral uptake without transgene expression (FIG. 5C). Thesetwo observations in CuFi and HEK293 monolayer cultures suggest thatpost-entry barriers, rather than receptor-mediated uptake, also play akey role in rAAV2/HBoV1 transduction.

To further investigate post-entry barriers to rAAV2/HBoV1 transduction,the influences of proteasome inhibitors were evaluated. Overall,proteasome inhibitors had little effect on viral uptake followinginfection under all the conditions evaluated (apical or basolateralinfection of polarized CuFi ALI or infection of non-polarized CuFimonolayers) (FIG. 5C). By contrast, proteasome inhibitor applicationduring the 4 hour infection period enhanced transduction 45-foldfollowing apical infection of polarized CuFi epithelia, while onlymarginally enhancing transduction following basolateral infection ofpolarized CuFi epithelia (5 fold) or infection of CuFi monolayers(4.3-fold) (FIG. 5C). These results suggest proteasome-dependentbarriers to intracellular processing of HBoV1 virions are greater fromthe apical membrane of polarized CuFi epithelia. Furthermore, CuFimonolayers appear to have the greatest post-entry block to HBoV1 virionprocessing that is also less proteasome-dependent. Cumulatively, theseresults suggest that polarization/differentiation of airway epithelialcells alter both receptor-mediated uptake and intracellular processingof HBoV1 virions.

A rAAV2/HBoV1 Vector Harboring a 5.5 kb Genome with a Strong CBA-hCFTRExpression Cassette can Correct CFTR-Mediated Chloride Currents in CFHAE.

The application of rAAV vectors for CF gene therapy has been hindered bythe relatively small packaging capacity of the virus and the large sizeof full-length CFTR cDNA (4443 bp of coding sequence). This hasnecessitated the use of very small synthetic weaker promoters and/or thedeletion of CFTR domains not critical for chloride channel function(Zhang et al., 1998). Both of these approaches are suboptimal forCFTR-mediated gene therapy. A rAAV2/HBoV1 vector would have enough spaceto accommodate strong transcription regulatory elements for human CFTRgene expression. To provide the proof-of-concept for this approach, arAAV2 proviral plasmid was constructed that harbored a 5.5 kb genomecontaining a 5.2 kb human CFTR expression cassette driven by the strongCBA promoter. When this proviral plasmid was packaged into HBoV1virions, the resultant viral yield (AV2/HBc.CBAhCFTR) averaged 1.5×10¹¹DRP from ten 150-mm dishes of transfected HEK293 cells, a similar levelof production for rAAV2/HBoV1 vectors with luciferase reporters. Theintegrity of the 5.5 kb rAAV genome within AV2/HBc.CBAhCFTR wasconfirmed by alkaline agarose gel analysis (data not shown).

To validate the function of AV2/HBc.CBAhCFTR virus, primarydifferentiated CF HAE were infected with 10¹⁰ DPR (5,000-10,000DRP/cell) from the apical surface in the presence of proteasomeinhibitors and assessed CFTR function at 10 days following infection.CFTR function was evaluated as the change in cAMP-mediated short-circuitcurrent (Isc) following stimulation with IBMX and forskolin andinhibition with GlyH101 (a CFTR inhibitor). DIDS and amiloride were usedto block non-CFTR chloride channels and ENaC-mediated sodium currentsprior to cAMP induction. Results comparing complementation ofCFTR-mediate chloride currents following apical infection withAV2/HBc.CF5tg83Luc (control vector) and AV2/HBc.CBAhCFTR are shown inFIG. 6A. A significant change in cAMP-inducible lsc was observedfollowing AV2/HBc.CBAhCFTR infection, as compared controlAV2/HBc.CF5tg83Luc infected samples, and this current was blocked by theaddition of the CFTR inhibitor GlyH101. FIG. 6B summarizes theΔlsc_((cAMP)) following cAMP agonist induction and the Δlsc_((glyH))following GlyH101 inhibition for the two infections conditions andnon-CF controls. The level of correction following AV2/HBc.CBAhCFTRinfection (Δlsc_((cAMP))=2.60+1-0.96 μA/cm² andΔlsc_((glyH))=2.98+/−0.73 μA/cm²) reflects about 30% of theΔlsc_((cAMP)) and Δlsc_((glyH)) observed in non-CF HAE. Expression ofhCFTR protein on the apical surface of AV2/HBc.CBAhCFTR infected CF HAEwas also confirmed by immunofluorescent staining (FIG. 6C). Littleimmunoreactivity was observed in the AV2/HBc.F5tg83Luc infected samples,as might be expected since ΔF508-CFTR is efficiently degraded in primaryCF HAE (Flotte, 2001).

Discussion

The newly discovered and partially characterized HBoV1 provides severalpotential attractive advantages for the design of CF airway gene therapyvectors. First, wild-type HBoV1 efficiently infects HAE from the apicalsurface at extremely low MOIs, suggesting that its capsid proteins arehighly adapted for airway infection. Second, wild-type HBoV1 has agenome of 5500-nt, suggesting that larger CFTR expression cassettescould be efficiently packaged into a recombinant HBoV1 virus. For thesereasons, we successfully generated both replication-defective rHBoV1 andpseudotyped rAAV2/HBoV1 vectors and studied their transduction profilesin HAE. Our findings demonstrate that rAAV2/HBoV1 vectors may indeed bean attractive alternative to rAAV vectors for gene therapy of CF.Additionally, our studies evaluating these new recombinant HBoV1-basedvectors have uncovered interesting biology regarding howpolarization/differentiation influences HBoV1 capsid-mediated infectionand transduction from the apical and basolateral membranes of HAE.

Although cross-genera parvovirus pseudopackaging has been known to bepossible for some time, the efficiency appears much higher forHBoV1-based vectors. For example, the efficiency of the rAAV2 genomeencapsidation in parvovirus B19 capsids yields viral titers of about 10⁹DRP/ml (Ponnazhagan et al., 1995), while yields of rAAV2/HBoV1 vectorsare about 2×10¹¹ DPR/ml. Yields of rAAV2/HBoV1 were slightly higher(about 2-4 fold) than that for rHBoV1, but similar to that of wild typeHBoV1 production in HEK 293 cells following transfection of theinfectious clone pIHBoV1 (Huang et al., 2012). However, it remains clearthat improvements in viral packaging are still needed, as the yield ofrAAV2/HBoV1 vectors remains about 10% of the level for rAAV2.

One unique aspect of the HBoV1 capsid is the fact it more efficientlytransduces (>100-fold) polarized airway epithelia from the apicalsurface as compared to the basolateral surface. This membrane polarityof HAE transduction is distinct from all other rAAV serotypes studied todate. For example, rAAV2, rAAV5, and rAAV6 preferentially transduce HAEfrom basolateral membrane with about 100-fold preference (Yan et al.,2013; Yan et al., 2006; Duan et al., 1998), while rAAV1 demonstrates anequal preference for transduction from both apical and basolateralmembranes (Yan et al., 2006). In the context of HBoV1 capsid,polarization appears to be key to induce viral receptors and/orco-receptors required for efficient transduction from the apicalmembrane. Indeed, enhanced viral genome uptake from the apical, ascompared to basolateral, membranes of polarized CuFi epithelia suggeststhat the expression of an HBoV1 receptor(s) is likely regulated bypolarization. The ability of proteasome inhibitors to effectivelyenhance rAAV2/HBoV1 transduction from the apical, but not basolateral,membrane also suggests that infection from these two membranes differswith respect to capsid processing biology of internalized HBoV1 virions.

Interestingly, the process of HBoV1 infection of non-polarized CuFicells represents a biologic process that is uniquely different from thatof apical or basolateral infection of polarized cells. In this context,CuFi monolayers exhibit efficient uptake of rAAV2/HBoV1, as seenfollowing apical infection of CuFi ALI cultures, but largely lackproteasome responsiveness as observed following basolateral infection ofCuFi ALI cultures. One potential explanation for these findings might bethe partitioning of certain binding receptor and co-receptor pairs atthe apical membrane that route virus to be productively processedthrough a proteasome-interacting pathway (FIG. 7). For example,following polarization, a binding receptor/co-receptor that efficientlyprocesses internalized virus may be shuttled to the apical membrane,resulting in low viral uptake from the basolateral membrane (FIG. 7A).In the case of CuFi monolayers, efficient binding receptors may remainon the surface and interact with a more abundant second co-receptor thatshuffles virus to an intracellular compartment that is less efficientfor transduction and non-responsive to proteasome inhibition (FIG. 7B).This second inefficient co-receptor may be sequestered in thebasolateral membrane of polarized cells, thus preventing interferencewith apical infection (FIG. 7A). This is only one scenario of many, andassumes the expression of binding receptors and co-receptors do notchange following polarization. Alternative explanations for thedifferences in transduction biology between the three CuFi models mayinvolve uniquely expressed binding receptors and/or co-receptors thatare influences by polarization and differentiation.

Like most rAAV serotypes, rAAV2/HBoV1 transduction of primary HAE fromthe apical membrane was significantly enhanced (>1000 fold) by theaddition of proteasome inhibitors at the time of apical infection (FIG.5B). Proteasome inhibitors have been shown to enhance trafficking ofrAAV virions to the nucleus by promoting ubiquitination of the capsid(Yan et al., 2002) and our results demonstrating no change inrAAV2/HBoV1 viral uptake following proteasome inhibitor treatment areconsistent with action at a post-entry point following infection.However, it remains unclear if the mechanism of the proteasome-sensitivepost-entry barrier is similar for rAAV and HBoV1 virions. For example,although we observed similar patterns of cytoplasmic and nucleardistribution between the rAAV2/HBoV1 and rAAV2/1 following apicalinfection of primary HAE, rAAV2/HBoV1 was about 10-fold more sensitiveto enhancement of transduction by proteasome inhibitors than previousobserved for rAAV1 (Yan et al., 2006). Differences in the mechanism ofvirion processing and uncoating between rAAVs and HBoV1 may beresponsible for these observations and warrants further investigation.

One of the most important differences between rAAV2/HBoV1 and rAAVvectors is the packaging capacity for a transgene cassette. rAAV2/HBoV1vectors can carry genomes up to 5.5 kb as compared to 4.9 kb for rAAVvectors. Although the upper limit of genome packaging within HBoV1capsids was not evaluated in this study, the 12% increase in genome size(600 bp) is very significant for delivery of CFTR expression cassettes.rAAV2/HBoV1 packaging enabled the use of a strong CBA promoter-drivenCFTR expression cassette, and resulted in very reasonable correction ofCFTR-dependent chloride currents in CF HAE. Based on the ability of rAAVto effectively package 5% more DNA than the wild type genome, it isreasonable to expect that HBoV1-based vectors may be capable ofefficiently packaging genomes up to 5.8 kb in length. Additionally, itmay be possible to encapsidate self-complementary (sc) double-strandedforms of rAAV genomes (2.7 to 2.8 kb in length) into HBoV1 capsids.

In conclusion, two new types of recombinant HBoV1-based vectors weredeveloped for efficient gene therapy to the human airway. However,chimeric rAAV2/HBoV1 vectors have three clear advantages over theauthentic rHBoV1 vectors for human gene therapy. First, rAAV2/HBoV1vector yields were significantly greater in an HEK293 cell productionsystem than that for rHBoV1. Second, the rAAV2 genome has already beenused in clinical trials and avoids potential safety concerns that mightaccompany use of a new viral genome. Third, the application of rHBoV1vectors could be hampered by the potential for contamination ofreplication-competent virus in the vector stocks, which could begenerated through homologous recombination of helper plasmids. Thislater concern is likely theoretical as we did not observe cytopathologyin HAE following infection with rHBoV1. Nonetheless, further vectordevelopment is required for the application of rHBoV1 to both minimizethe potential for replication-competent virus and improve vector yields.

Despite the promise of this new rAAV2/HBoV1 vector system, severalunknowns require further investigation prior to considering clinicalapplications. For example, is there pre-existing airway humoral immunityto HBoV1 caspids in most CF patients, and if so, does this impactrAAV2/HBoV1 infection? Studies have suggested that approximately 71% ofhumans ranging from birth to 41 years of age contain circulatingantibodies against HBoVs (Schildgren et al., 2005), however, nothing isknown concerning neutralizing antibodies to this virus in the airway.Given that HBoV1 infections primarily occur within the first year oflife, it is assumed that such immunity is protective to secondaryinfections. However, the frequent detection of HBoVs in stool from bothhealthy children and adults, as well as seroepidemiology studies,suggests that viral shedding can occur for long periods and/or patientscan have frequent recurrent infections (Kapoor et al., 2010). Secondaryinfections or anamnestic immune responses also appear to commonly occur,and while HBoV1 primary infections are strongly associated withrespiratory illness, secondary immuno-activation by HBoV1 is not(Meriluoto et al., 2012). It remains unclear whether such humoralimmunity can prevent infection from the airway surface or acts to limitreplication and spread of HBoV1 (Korner et al., 2011). The fact thatwild type HBoV1 shows long-term and low-level persistence in therespiratory tract following primary infection (Martin et al., 2010;Allander et al., 2005) suggests that this virus may have methods ofevading immune detection. The development of rHBoV1 and rAAV2/HBoV1vectors should enable such questions to be addressed using HAEreconstitution experiments combining recombinant reporter virus withserum or bronchioalveolar lavage samples from HBoV1 infected patients.Despite these unknowns, the development of HBoV1-based recombinantvectors may have unique utilities for CF gene therapy and/or vaccinationof infants to protect from wild type HBoV1 infections.

Interestingly, the process of HBoV1 infection of non-polarized CuFicells represents a biologic process that is uniquely different from thatof apical or basolateral infection of polarized cells. In this context,CuFi monolayers exhibit efficient uptake of rAAV2/HBoV1, as seenfollowing apical infection of CuFi ALI cultures, but largely lackproteasome responsiveness as observed following basolateral infection ofCuFi ALI cultures. One potential explanation for these findings might bethe partitioning of certain binding receptor and co-receptor pairs atthe apical membrane that route virus to be productively processedthrough a proteasome-interacting pathway (FIG. 7). For example,following polarization, a binding receptor/co-receptor that efficientlyprocesses internalized virus may be shuttled to the apical membrane,resulting in low viral uptake from the basolateral membrane (FIG. 7A).In the case of CuFi monolayers, efficient binding receptors may remainon the surface and interact with a more abundant second co-receptor thatshuttles virus to an intracellular compartment that is less efficientfor transduction and non-responsive to proteasome inhibition (FIG. 7B).This second inefficient co-receptor may be sequestered in thebasolateral membrane of polarized cells, thus preventing interferencewith apical infection (FIG. 7A). This is only one scenario of many andassumes the expression of binding receptors and co-receptors do notchange following polarization. Alternative explanations for thedifferences in transduction biology between the three CuFi models mayinvolve uniquely expressed binding receptors and/or co-receptors thatare influences by polarization and differentiation.

Like most rAAV serotypes. rAAV2/HBoV1 transduction of primary HAE fromthe apical membrane was significantly enhanced (>1000 fold) by theaddition of proteasome inhibitors at the time of apical infection (FIG.5B). Proteasome inhibitors have been shown to enhance trafficking ofrAAV virions to the nucleus by promoting ubiquitination of the capsid(Yan et al., 2002) and our results demonstrating no change inrAAV2/HBoV1 viral uptake following proteasome inhibitor treatment areconsistent with action at a post-entry point following infection.However, it remains unclear if the mechanism of the proteasome-sensitivepost-entry barrier is similar for rAAV and HBoV1 virions. For example,although similar patterns of cytoplasmic and nuclear distribution wereobserved between the rAAV2/HBoV1 and rAAV2/1 following apical infectionof primary HAE, rAAV2/HBoV1 was about 10-fold more sensitive toenhancement of transduction by proteasome inhibitors than previousobserved for rAAV1 (Yan et al., 2006). Differences in the mechanism ofvirion processing and uncoating between rAAVs and HBoV1 may beresponsible for these observations and warrants further investigation.

One of the most important differences between rAAV2/HBoV1 and rAAVvectors is the packaging capacity for a transgene cassette. rAAV2/HBoV1vectors can carry genomes up to 5.5 kb as compared to 4.9 kb for rAAVvectors. Although the upper limit of genome packaging within HBoV1capsids was not evaluated in this study, the 12% increase in genome size(600 bp) is very significant for delivery of CFTR expression cassettes.rAAV2/HBoV1 packaging enabled the use of a strong CBA promoter-drivenCFTR expression cassette, and resulted in very reasonable correction ofCFTR-dependent chloride currents in CF HAE. Based on the ability of rAAVto effectively package about 5% more DNA than the wild type genome, itis reasonable to expect that HBoV1-based vectors may be capable ofefficiently packaging genomes up to 5.8 kb in length.

In conclusion, two new types of recombinant HBoV1-based vectors weredeveloped for efficient gene therapy to the human airway. However,chimeric rAAV2/HBoV1 vectors have three clear advantages over theauthentic rHBoV1 vectors for human gene therapy. First, rAAV2/HBoV1vector yields were significantly greater in an HEK293 cell productionsystem than that for rHBoV1. Second, the rAAV2 genome has already beenused in clinical trials and avoids potential safety concerns that mightaccompany use of a new viral genome. Third, the application of rHBoV1vectors could be hampered by the potential for contamination ofreplication-competent virus in the vector stocks, which could begenerated through homologous recombination of helper plasmids. Thislater concern is likely theoretical as cytopathology in HAE was notobserved following infection with rHBoV1.

Example 4

An optimized HBoV1 capsid helper, pCMVHBoV1 NS1(−)Cap (FIG. 10A), inwhich a strong CMV promoter was used and the NS1 ORF was terminatedearly, yielded a significant increase in production (to about 1.5×10¹²DRP/20 145-mm plates of transfected HEK293 cells), a level comparable tothat for rAAV2/2 (FIG. 9C). As rAAV vector production in Sf9 insectcells with baculovirus expression vector (BEV) is highly efficient andlinearly scalable, a BEV-based rAAV2/HBoV1 vector production system wasestablished to further increase yield. Specifically, the following BEVvectors were constructed (FIG. 10A): AAV2 Rep helper baculovirus(Bac-Rep), which expresses AAV2 Rep78/Rep52; HBoV1 Cap helper virus(Bac-Cap), which expresses HBoV1 capsid proteins VP1, VPx, and VP2; andtransfer vector (Bac-rAAV), which contains an rAAV2 genome. Theexpression of these vectors and their ability to promote replication ofthe rAAV2 genome were confirmed (FIG. 10B). Co-infection of 200 mL ofSf9 cell culture (2×10⁶ cells/mL) with an equal MOI (5 pfu/cell) of eachvirus produced vector at a yield of 1×10¹² DRP (FIGS. 10C&D), and thevectors produced from Sf9 and 293 cells had a similar ability totransduce CuFi-ALI (FIG. 10E). These results demonstrate that infectiousrAAV2/HBoV1 vector can be produced in the BEV-Sf9 cell system asefficiently as in the optimized 4-plasmid co-transfected-293 cell system(FIG. 9). Even before optimization, the production of rAAV2/HBoV1 vectorfrom Sf9 cells achieved 10% of the yield of rAAV2/2 vector from Sf9cells (FIG. 10D). As rAAV vector production in Sf9 cells can be scaledup using a bioreactor and further increased using a rep- andcap-expressing Sf9 cell line, it was hypothesized that rAAV2/HBoV1production in a bioreactor can be increased a further ten fold, to ayield above 10¹⁴ DRP per liter of Sf9 cell culture.

Increased packaging capability is one of the key advantages of therAAV2/HBoV1 vector, especially with respect to regulating expression ofthe CFTR gene in the HAE. Although an oversized rAAV2 genome (5.5 kb)can be encapsidated in the HBoV1 capsid to enable effective expressionof FL-CFTR ORF under the control of a strong CBA promoter, expansion bya further 5% would make it possible to utilize the FOXJ1 promoter, whichdrives expression in the ciliated cells of the airway epithelium(Verdiev & Descoates, 1999), and to also incorporate most of the keysequences of the CFTR 3′UTR (microRNA-targeting sites) into the rAAV2genome for appropriate regulation of CFTR expression.

The HBoV1 capsid packaged a larger rAAV2 genome of 5.9 kb without asignificant loss vector production (FIG. 11). And as shown in FIG. 12,the rAAV2/HBoV1 vector efficiently transduced ciliated and K18-positiveepithelial cells in HAE-ALI cultures.

Example 5

rAAV2/HBoV1 Vectors Encoding a Human Full-Length CFTR ORF.

Due to limitations of the packaging capacity of rAAV, an 83-bp syntheticpromoter (tg83) was used to drive expression of the FL-CFTR ORF inAV2/2.tg83-CFTR (FIG. 13B). A screen of a synthetic oligonucleotide(about 100 bp) library of >50,000 unique sequences (Schalabach et al.,2010) for short enhancers revealed that the F5 enhancer raises tg83promoter activity to a level as high as 60% of that of the strong CMVpromoter in HAE-ALI (FIG. 13A). This promoter was engineered into theAV2/HBc.F5tg83luc and AV2.HBc.F5tg83luc-CMVmcherry (FIG. 12) vectors.Incorporation of the short (185-nt) but efficient F5tg83 promoter forCFTR expression in rAAV2/2 vectors necessitates the use of a shortenedCFTR ORF, for example a partial deletion (159 bp) at the R-domain[CFTR(ΔR)] (Ostedgaard et al., 2002; Gillen et al., 2011), which mightcompromise CFTR function (FIG. 13B) besides the natural (albeit low)apical tropism of the AAV2/2 vector. However, the F5tg83 could serve asan ideal promoter for driving expression of the FL-CFTR ORF in therAAV2/HBoV1 vector. The AV2/HBc.F5tg83hCFTR vector has at least 600 bpof space for further optimization of regulatory CFTR expression. Byincorporating a short CFTR 3′UTR sequence that contains targeting sitesfor microRNAs that have been reported to regulate CFTR expression, e.g.,miR101, miR-145 and miR-494 (Gillen et al., 2011; Megiorni et al.,2011), the (post-)transcriptional regulation strategy will enableautonomous regulation of CFTR expression, and thus bring aboutendogenous levels of CFTR expression and more physiologiccomplementation patterns To incorporate longer regulatory elements, suchas a 1-kb ciliated cell-specific promoter, FOXJ1 (Ostrowski et al.,2003) for site-specific CFTR expression from the AV2/HBc.FOXJ1hCFTRvector of 5.8 kb (FIG. 13C), needs an expandable package capacity ofHBoV1 capsid.

To generate new rAAV2 constructs to be pseudotyped in the HBoV1 capsid(AV2.F5tg83hCFTR, AV2.F5tg83hCFTR(plus) and AV2.FOXJ1hCFTR) (FIG. 13C)and to compare their effectiveness in correcting the CFTR-specific Cl⁻defect with AV2/HBc.CBAhCFTR, the functionalities of the new vectors inCuFi-ALI derived from the immortalized human CF airway cell line CuFi8(genotype: ΔF508/ΔF508) are tested. The changes of short circuit current(Isc) for the complementation of cAMP-regulated Cl⁻ channel activitiesare measured, and the levels of fully processed CFTR protein on theapical surface at 10 days p.i. examined. To test the hypothesis that theexpression of CFTR at a more physiologic level will restore CFTRactivity, side-by side comparative assessments of CFTR expression levelsand function in CF xenografts infected using these vectors with fourvector doses spanning a 50-fold range is conducted. Transepithelialpotential differences (TEPDs) are measured to assess the level of CFTRcomplementation. Then airway fluid is harvested for in vitro bacterialkilling assays, as well as in vivo bacterial challenge experimentsassessing bacterial clearance (at termination of the experiment).Expression of CFTR on the surface epithelium will be quantified by IFstaining at the apical membrane, using Metamorph software and/orimmunoprecipitation kinase assays for the fully-processed B and C.Vector-derived CFTR mRNA and the number of intracellular vector genomecopies (vgc) in graft samples will be quantified by qPCR, as describedby our laboratory previously.

Most of the CFTR mutations that are associated with severe CF lungdisease are located downstream of exon 10 (FIG. 14C). The SMaRT approachwould repair CFTR malfunction at the mRNA level, and would affect onlythose cells that express CFTR endogenously. This repair technique relieson hybridization of intronic domains in the vector-derived pre-mRNA andthe endogenous CFTR pre-mRNA to trigger a trans-splicing process thatreconstitutes a mutation-free CFTR mRNA. Vector AV2.CMV-PTM24CF (FIG.14A) encodes a pre-therapeutic RNA molecule (PTM), which consists of anoptimized trans-spicing domain (FIG. 14B) and CFTR exons 10-26. Thetrans-spicing domain targets a PTM24-complementary/binding RNA sequence(binding domain=BD) near the 3′ splice site (SS) of intron 9, resultingin trans-splicing from the 5′SS of intron 9 of the endogenous(defective) CFTR pre-mRNA to the 3′SS of the PTM RNA, and subsequentlyto production of a functional CFTR mRNA (FIG. 14C). The effectiveness ofthe PTM in AV2.CMV-PTM24CF has been validated in CF (ΔF508/ΔF508)HAE-ALI (Ostrowski et al., 2003). The high efficiency of apicaltransduction and the expanded genome capacity of the rAAV2/HBoV1 vectormay overcome the problem of the inherent weakness of the rAAV vectorwith respect to correcting CFTR expression through the SMaRT approach,and that a fully-reconstituted CFTR mRNA with an intact 3′UTR (1,557 bp)(Ostrowski et al., 2003) provides precisely regulated CFTR proteinexpression at endogenous level through post-transcriptional regulation,e.g., regulation by miRNAs.

The effectiveness of a 5.55 kb rAAV2 PTM genome, AV2.CBA-PTM24CF-3UTR,in rescuing CFTR function is tested (FIG. 14D). While retaining thealready optimized PTM trans-splicing domain from the first-generationvector (AV2.CMV-PTM24CF), this genome also includes a 1.5 kb 3′UTR ofthe CFTR cDNA and use the CBA promoter instead of the CMV promoter.AV2.CBA-PTM24CF, which does not transcribe the 3′UTR, will serve as acontrol. The rAAV2 genomes AV2.CBA-PTM24CF-3UTR and AV2.CBA-PTM24CF arepseudotyped, using the HBoV1 capsid for its high packaging capacity andefficient apical transduction. These rAAV2/HBoV1 PTM vectors areapically applied to CF HAE-ALI cultures at an MOI of 5 k, and thefunctional correction of CFTR expression will be assessed at 3 days and1 week p.i. At 4 weeks p.i., the experiment will be terminated and invivo bacterial challenge assays will be conducted to assess bacterialclearance in the grafts.

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification, thisinvention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details herein may be varied considerably without departing fromthe basic principles of the invention.

1-6. (canceled)
 7. A method to express a heterologous gene product inmammalian cells comprising providing isolated chimeric virus comprisingbocavirus (BoV) capsid protein and a recombinant adeno-associated viral(AAV) genome comprising an expression cassette encoding the heterologousgene product; and infecting the cells with the virus in an amounteffective to express the heterologous gene product.
 8. The method ofclaim 7 wherein the gene product is a therapeutic gene product, acatalytic RNA, a microRNA, RNA pre-transplicing molecule (PTM-RNA), aneutralizing antibody or an antigen binding fragment thereof, aprophylactic gene product, a polypeptide or a peptide.
 9. The method ofclaim 7 further comprising contacting the mammalian cells with at leastone agent in an amount effective to additively or synergisticallyenhance rAAV transduction. 10-11. (canceled)
 12. A method to inhibit ortreat a condition associated with aberrant expression of an endogenousgene product, comprising: contacting a mammal at risk of or having thecondition, with an effective amount of an isolated chimeric viruscomprising bocavirus capsid proteins and a rAAV genome, wherein the rAAVgenome comprises a transgene encoding at least a portion of atherapeutic gene product, the expression of which in the mammal inhibitsor treats at least one symptom of the condition.
 13. The method of claim12 wherein the transgene encodes cystic fibrosis transmembraneconductance regulator, β-globin, γ-globin, alpha-antitrypsin, tyrosinehydroxylase, glucocerebrosidase, aryl sulfatase A, factor VIII,dystrophin or erythropoietin.
 14. (canceled)
 15. The method of claim 12further comprising administering an agent that enhances AAV transductioncomprising a lipid lowering agent, a mucolytic agent, a food additive,LLnL, Z-LLL, bortezomib (Velcade), epoxomicin, doxorubicin, doxil,daunorubicin, idanibicin epirubicin, aclarubicin, simvastatin, tannicacid, camptothecin, or cisplatin. 16-19. (canceled)
 20. The method ofclaim 9 wherein the cell or mammal is contacted with the agent beforethe cell or mammal is contacted with the virus.
 21. The method of claim9 wherein the cell or mammal is contacted with the virus before the cellor mammal is contacted with the agent.
 22. (canceled)
 23. The method ofclaim 15 wherein the agent and the virus are administered to the lung,nasal epithelium, gastrointestinal tract, or blood.
 24. (canceled)
 25. Amethod to immunize a mammal, comprising: administering to a mammal anisolated chimeric virus comprising bocavirus capsid proteins and a rAAVgenome encoding a prophylactic gene product in an amount effective toprevent or inhibit microbial infection or replication.
 26. The method ofclaim 25 wherein the gene product is an antigen of a virus or bacteria.27-48. (canceled)
 49. The method of claim 26 wherein the antigen is abocavirus antigen.
 50. The method of claim 49 wherein the chimeric virusis administered to the lung, gastrointestinal tract, nasal epithelium orblood. 51-58. (canceled)
 59. The method of claim 7 wherein the geneproduct encodes a therapeutic protein.
 60. The method of claim 7 whereinthe rAAV genome is a r AAV-1, rAAV-2, r AAV-3, rAAV-4, rAAV-5, rAAV-6,rAAV-7, rAAV-8 or rAAV-9 genome.
 61. The method of claim 7 wherein thegene product is a viral antigen, bacterial antigen, tumor antigen,parasite antigen, or fungal antigen.
 62. The method of claim 7 whereinthe gene product is cystic fibrosis transmembrane conductance regulator,b-globin, g-globin, tyrosine hydroxylase, glucocerebrosidase, arylsulfatase A, factor VIII, dystrophin, alpha 1-antitrypsin, surfactantprotein SP-D, SP-A or SP-C, erythropoietin, HBoV protein, influenzavirus protein, respiratory syncytial virus (RSV) protein, a neutralizingantibody or an antigen binding fragment thereof, severe acuterespiratory syndrome (SARS) virus protein, or a cytokine selected fromthe group consisting of interferon (IFN)-alpha, IFN-gamma, tumornecrosis factor (TNF), interleukin (IL)-1, IL-17, or IL-6.
 63. Themethod of claim 7 wherein the HBoV is HBoV1.
 64. The method of claim 7wherein the BoV is HBoV2.
 65. The method of claim 7 wherein the BoV isHBoV3.
 66. The method of claim 7 wherein the BoV is HBoV4.
 67. Themethod of claim 7 wherein the heterologous gene product comprises acystic fibrosis transmembrane conductance regulator.